Novel specific-binding polypeptides and uses thereof

ABSTRACT

The present invention relates to novel, specific-binding therapeutic and/or diagnostic polypeptides directed against the target of Swiss Prot Q16552 and novel, specific-binding therapeutic and/or diagnostic polypeptides directed against the target of Swiss Prot Q9NPF7. In addition, the present invention relates to novel, specific-binding therapeutic and/or diagnostic polypeptides directed against one or both of Swiss Prot Q16552 and Swiss Prot Q9NPF7. The invention also relates to nucleic acid molecules encoding such polypeptides and to methods for generation of such polypeptides and nucleic acid molecules. In addition, the invention is directed to compositions comprising the polypeptides, and therapeutic and/or diagnostic uses of these polypeptides.

I. BACKGROUND

Muteins of various lipocalins are a rapidly expanding class oftherapeutics. Indeed, lipocalin muteins can be constructed to exhibit ahigh affinity and specificity against a target that is different than anatural ligand of wild type lipocalins (e.g., WO 99/16873, WO 00/75308,WO 03/029463, WO 03/029471 and WO 05/19256), such as Interleukin-17A orInterleukin-23.

A. Interleukin-17A

Interleukin-17A (IL-17A, synonymous with IL-17) is a cytokine producedfrom the Th17 lineage of T cells. IL-17 was originally designated“CTL-associated antigen 8” (CTLA-8) (Rouvier et al., J. Immunol, 1505445-5556 (1993); Yao et al., Immunity, 3: 811-821 (1995)). The humanequivalent of CTLA-8 was later cloned and designated “IL-17” (Yao etal., J. Immunol, 155(12): 5483-5486 (1995); Fossiez et al., J. Exp.Med., 183(6): 2593-2603 (1996)).

Human IL-17A (CTLA-8, further named as IL-17, Swiss Prot Q16552) is aglycoprotein with a Mr of 17,000 daltons (Spriggs et al., J. Clin.Immunol, 17: 366-369 (1997)). IL-17A may exist as either a homodimerIL-17 A/A or as a heterodimer complexed with the homolog IL-17F to formheterodimeric IL-17 A/F. IL-17F (IL-24, ML-1) shares a 55% amino acididentity with IL-17A. IL-17A and IL-17F also share the same receptor(IL-17RA), which is expressed on a wide variety of cells includingvascular endothelial cells, peripheral T cells, B cells, fibroblast,lung cells, myelomonocytic cells, and marrow stromal cells (Kolls etal., Immunity, 21: 467-476 (2004); Kawaguchi et al., J. Allergy Clin.Immunol, 114(6): 1267-1273 (2004); Moseley et al., Cytokine GrowthFactor Rev., 14(2): 155-174 (2003)). Additional IL-17 homologs have beenidentified (IL-17B, IL-17C, IL-17D, and IL-17E). These other familymembers share less than 30% amino acid identity with IL-17A (Kolls etal., 2004).

IL-17A is mainly expressed by Th17 cells and is present at elevatedlevels in synovial fluid of patients with rheumatoid arthritis (RA) andhas been shown to be involved in early RA development. IL-17A is alsoover-expressed in the cerebrospinal fluid of multiple sclerosis (MS)patients. In addition, IL-17 is an inducer of TNF-α and IL-1, the latterbeing mainly responsible for bone erosion and the very painfulconsequences for affected patients (Lubberts E. (2008) Cytokine, 41, p.84-91). Furthermore, inappropriate or excessive production of IL-17A isassociated with the pathology of various other diseases and disorders,such as osteoarthritis, loosening of bone implants, acute transplantrejection (Antonysamy et al., (1999) J. Immunol, 162, p. 577-584; vanKooten et al. (1998) J. Am. Soc. Nephrol., 9, p.1526-1534), septicemia,septic or endotoxic shock, allergies, asthma (Molet et al., (2001) J.Allergy Clin. Immunol., 108, p. 430-438), bone loss, psoriasis(Teunissen et al. (1998) J. Invest. Dermatol, 111, p. 645-649),ischemia, systemic sclerosis (Kurasawa et al., (2000) Arthritis Rheum.,43, p. 2455-2463), stroke, and other inflammatory disorders.

Although a variety of inhibitors of IL-17A have been described, sincethe discovery of this critical proinflammatory cytokine, currentapproaches are not optimal, such as the necessity of complex mammaliancell production systems, a dependency on disulfide bond stability, thetendency of some antibody fragments to aggregate, limited solubility andlast but not least, they may elicit undesired immune responses even whenhumanized. There remains a need, therefore, to develop proteins such aslipocalin muteins with binding-affinity for IL-17A.

B. Interleukin-23

Interleukin-23 (also known as IL-23) is a heterodimeric cytokinecomprised of two subunits, i.e., p19 and p40 (B. Oppmann et al, Immunity13, 715 (2000)). The p19 (Swiss Prot Q9NPF7, herein referred tointerchangeably as “IL-23p19”) subunit is structurally related to IL-6,granulocyte-colony stimulating factor (G-CSF), and the p35 subunit ofIL-12. IL-23 mediates signaling by binding to a heterodimeric receptor,comprised of IL-23R and IL-12beta1. The IL-12beta1 subunit is shared bythe IL-12 receptor, which is composed of IL-12beta1 and IL-12beta2.Transgenic p19 mice have been recently described to display profoundsystemic inflammation and neutrophilia (M. T. Wiekowski et al, J Immunol166, 7563 (2001)).

Human IL-23 has been reported to promote the proliferation of T cells,in particular memory T cells and can contribute to the differentiationand/or maintenance of Thl 7 cells (D. M. Frucht, Sci STKE 2002 Jan. 8;2002(114):PE1).

Although a variety of selective inhibitors of IL-23 (via binding to thep19 subunit) have been described, since the discovery of this criticalheterodimeric cytokine, these current approaches still have a number ofserious drawbacks, such as the necessity of complex mammalian cellproduction systems, a dependency on disulfide bond stability, thetendency of some antibody fragments to aggregate, limited solubility andlast but not least, they may elicit undesired immune responses even whenhumanized. There is an unmet need to, therefore, to develop proteinssuch as lipocalin muteins with binding-affinity for IL-23.

II. DEFINITIONS

The following list defines terms, phrases, and abbreviations usedthroughout the instant specification. All terms listed and definedherein are intended to encompass all grammatical forms.

As used herein, “IL-17A” (including IL-17 A/A as well as IL-17A incomplex with IL-17F, also termed as IL-17 A/F) means a full-lengthprotein defined by Swiss Prot Q16552, a fragment thereof, or a variantthereof.

As used herein, “IL-23p19” means a full-length protein defined by SwissProt Q9NPF7, a fragment thereof, or a variant thereof.

As used herein, “detectable affinity” means the ability to bind to aselected target with an affinity constant of generally at least about10⁻⁵ M. Lower affinities are generally no longer measurable with commonmethods such as ELISA and therefore of secondary importance. Forexample, binding affinities of lipocalin muteins according to thedisclosure may in some embodiments be of a K_(D) below 800 nM, in someembodiments be of a K_(D) below 30 nM and in some embodiments about 50picomolar (pM) or below.

As used herein, “binding affinity” of a protein of the disclosure (e.g.a mutein of a lipocalin) or a fusion protein thereof to a selectedtarget (in the present case, IL-17A or IL-23p19), can be measured (andthereby KD values of a mutein-ligand complex be determined) by amultitude of methods known to those skilled in the art. Such methodsinclude, but are not limited to, fluorescence titration, competitionELISA, calorimetric methods, such as isothermal titration calorimetry(ITC), and surface plasmon resonance (BIAcore). Such methods are wellestablished in the art and examples thereof are also detailed below.

It is also noted that the complex formation between the respectivebinder and its ligand is influenced by many different factors such asthe concentrations of the respective binding partners, the presence ofcompetitors, pH and the ionic strength of the buffer system used, andthe experimental method used for determination of the dissociationconstant K_(D) (for example fluorescence titration, competition ELISA orsurface plasmon resonance, just to name a few) or even the mathematicalalgorithm which is used for evaluation of the experimental data.

Therefore, it is also clear to the skilled person that the K_(D) values(dissociation constant of the complex formed between the respectivebinder and its target/ligand) may vary within a certain experimentalrange, depending on the method and experimental setup that is used fordetermining the affinity of a particular lipocalin mutein for a givenligand. This means that there may be a slight deviation in the measuredK_(D) values or a tolerance range depending, for example, on whether theK_(D) value was determined by surface plasmon resonance (Biacore), bycompetition ELISA, or by “direct ELISA.”

As used herein, a “mutein,” a “mutated” entity (whether protein ornucleic acid), or “mutant” refers to the exchange, deletion, orinsertion of one or more nucleotides or amino acids, compared to thenaturally occurring (wild-type) nucleic acid or protein “reference”scaffold.

The term “fragment” as used herein in connection with the muteins of thedisclosure relates to proteins or peptides derived from full-lengthmature human tear lipocalin that are N-terminally and/or C-terminallyshortened, i.e. lacking at least one of the N-terminal and/or C-terminalamino acids. Such fragments may include at least 10, more such as 20 or30 or more consecutive amino acids of the primary sequence of the maturelipocalin and are usually detectable in an immunoassay of the maturelipocalin. Said term also includes fragments of a mutein and variants asdescribed herein. Lipocalin muteins of the present invention, fragmentsor variants thereof preferably retain the function of binding to IL-17Aand/or IL23p19 as described herein.

In general, the term “fragment”, as used herein with respect to thecorresponding protein ligand IL-17A (including IL-17 A/A and IL-17 A/F)or IL-23p19 of a lipocalin mutein of the disclosure or of thecombination according to the disclosure or of a fusion protein describedherein, relates to N-terminally and/or C-terminally shortened protein orpeptide ligands, which retain the capability of the full length ligandto be recognized and/or bound by a mutein according to the disclosure.

The term “mutagenesis” as used herein means that the experimentalconditions are chosen such that the amino acid naturally occurring at agiven sequence position of the mature lipocalin can be substituted by atleast one amino acid that is not present at this specific position inthe respective natural polypeptide sequence. The term “mutagenesis” alsoincludes the (additional) modification of the length of sequencesegments by deletion or insertion of one or more amino acids. Thus, itis within the scope of the disclosure that, for example, one amino acidat a chosen sequence position is replaced by a stretch of three randommutations, leading to an insertion of two amino acid residues comparedto the length of the respective segment of the wild type protein. Suchan insertion of deletion may be introduced independently from each otherin any of the peptide segments that can be subjected to mutagenesis inthe disclosure. In one exemplary embodiment of the disclosure, aninsertion of several mutations may be introduced into the loop AB of thechosen lipocalin scaffold (cf. International Patent Application WO2005/019256 which is incorporated by reference its entirety herein).

The term “random mutagenesis” means that no predetermined single aminoacid (mutation) is present at a certain sequence position but that atleast two amino acids can be incorporated with a certain probability ata predefined sequence position during mutagenesis.

“Identity” is a property of sequences that measures their similarity orrelationship. The term “sequence identity” or “identity” as used in thepresent disclosure means the percentage of pair-wise identicalresidues—following (homologous) alignment of a sequence of a polypeptideof the disclosure with a sequence in question—with respect to the numberof residues in the longer of these two sequences. Identity is measuredby dividing the number of identical residues by the total number ofresidues and multiplying the product by 100.

The term “homology” is used herein in its usual meaning and includesidentical amino acids as well as amino acids which are regarded to beconservative substitutions (for example, exchange of a glutamate residueby an aspartate residue) at equivalent positions in the linear aminoacid sequence of a polypeptide of the disclosure (e.g., any lipocalinmutein of the disclosure).

The percentage of sequence homology or sequence identity can, forexample, be determined herein using the program BLASTP, version blastp2.2.5 (Nov. 16, 2002; cf. Altschul, S. F. et al. (1997) Nucl. Acids Res.25, 3389-3402). In this embodiment the percentage of homology is basedon the alignment of the entire polypeptide sequences (matrix: BLOSUM 62;gap costs: 11.1; cutoff value set to 10⁻³) including the propeptidesequences, preferably using the wild type protein scaffold as referencein a pairwise comparison. It is calculated as the percentage of numbersof “positives” (homologous amino acids) indicated as result in theBLASTP program output divided by the total number of amino acidsselected by the program for the alignment.

Specifically, in order to determine whether an amino acid residue of theamino acid sequence of a lipocalin (mutein) different from a wild-typelipocalin corresponds to a certain position in the amino acid sequenceof a wild-type lipocalin, a skilled artisan can use means and methodswell-known in the art, e.g., alignments, either manually or by usingcomputer programs such as BLAST2.0, which stands for Basic LocalAlignment Search Tool or ClustalW or any other suitable program which issuitable to generate sequence alignments. Accordingly, a wild-typelipocalin can serve as “subject sequence” or “reference sequence”, whilethe amino acid sequence of a lipocalin different from the wild-typelipocalin described herein serves as “query sequence”. The terms“reference sequence” and “wild type sequence” are used interchangeablyherein.

“Gaps” are spaces in an alignment that are the result of additions ordeletions of amino acids. Thus, two copies of exactly the same sequencehave 100% identity, but sequences that are less highly conserved, andhave deletions, additions, or replacements, may have a lower degree ofidentity. Those skilled in the art will recognize that several computerprograms are available for determining sequence identity using standardparameters, for example Blast (Altschul, et al. (1997) Nucleic AcidsRes. 25, 3389-3402), Blast2 (Altschul, et al. (1990) J. Mol. Biol. 215,403-410), and Smith-Waterman (Smith, et al. (1981) J. Mol. Biol. 147,195-197).

The term “variant” as used in the present disclosure relates toderivatives of a protein or peptide that include modifications of theamino acid sequence, for example by substitution, deletion, insertion orchemical modification. Such modifications do in some embodiments notreduce the functionality of the protein or peptide. Such variantsinclude proteins, wherein one or more amino acids have been replaced bytheir respective D-stereoisomers or by amino acids other than thenaturally occurring 20 amino acids, such as, for example, ornithine,hydroxyproline, citrulline, homoserine, hydroxylysine, norvaline.However, such substitutions may also be conservative, i.e. an amino acidresidue is replaced with a chemically similar amino acid residue.Examples of conservative substitutions are the replacements among themembers of the following groups: 1) alanine, serine, and threonine; 2)aspartic acid and glutamic acid; 3) asparagine and glutamine; 4)arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and6) phenylalanine, tyrosine, and tryptophan. The term “variant”, as usedherein with respect to the corresponding protein ligand IL-17A(including IL-17 A/A and IL-17 A/F) or IL-23p19 of a lipocalin mutein ofthe disclosure or of the combination according to the disclosure or of afusion protein described herein, relates to an IL-17 protein or fragmentthereof or IL-23 protein or fragment thereof, respectively, that has oneor more such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 40, 50, 60, 70, 80 or more amino acid substitutions,deletions and/or insertions in comparison to a wild-type IL-17A orIL-23p19 protein, respectively, such as an IL-17A or IL-23p19 referenceprotein as deposited with SwissProt as described herein. AN IL-17A orIL-23p19 variant, respectively, has preferably an amino acid identity ofat least 50%, 60%, 70%, 80%, 85%, 90% or 95% with a wild-type IL-17A orIL-23p19 protein, respectively, such as an IL-17A or IL-23p19 referenceprotein as deposited with SwissProt as described herein.

By a “native sequence” lipocalin is meant a lipocalin that has the sameamino acid sequence as the corresponding polypeptide derived fromnature. Thus, a native sequence lipocalin can have the amino acidsequence of the respective naturally-occurring lipocalin from anyorganism, in particular a mammal. Such native sequence polypeptide canbe isolated from nature or can be produced by recombinant or syntheticmeans. The term “native sequence” polypeptide specifically encompassesnaturally-occurring truncated or secreted forms of the lipocalin,naturally-occurring variant forms such as alternatively spliced formsand naturally-occurring allelic variants of the lipocalin. A polypeptide“variant” means a biologically active polypeptide having at least about50%, 60%, 70%, 80% or at least about 85% amino acid sequence identitywith the native sequence polypeptide. Such variants include, forinstance, polypeptides in which one or more amino acid residues areadded or deleted at the N- or C-terminus of the polypeptide. Generally avariant has at least about 70%, including at least about 80%, such as atleast about 85% amino acid sequence identity, including at least about90% amino acid sequence identity or at least about 95% amino acidsequence identity with the native sequence polypeptide. As anillustrative example, the first 4 N-terminal amino acid residues (HHLA)and the last 2 C-terminal amino acid residues (Ser, Asp) can be deleted,for example, in a tear lipocalin (Tlc) mutein of the disclosure withoutaffecting the biological function of the protein, e.g. SEQ ID NO: 1.

The term “position” when used in accordance with the disclosure meansthe position of either an amino acid within an amino acid sequencedepicted herein or the position of a nucleotide within a nucleic acidsequence depicted herein. To understand the term “correspond” or“corresponding” as used herein in the context of the amino acid sequencepositions of one or more lipocalin muteins, a corresponding position isnot only determined by the number of the preceding nucleotides/aminoacids. Accordingly, the position of a given amino acid in accordancewith the disclosure which may be substituted may vary due to deletion oraddition of amino acids elsewhere in a (mutant or wild-type) lipocalin.Similarly, the position of a given nucleotide in accordance with thepresent disclosure which may be substituted may vary due to deletions oradditional nucleotides elsewhere in a mutein or wild type lipocalin5-untranslated region (UTR) including the promoter and/or any otherregulatory sequences or gene (including exons and introns).

Thus, for a corresponding position in accordance with the disclosure, itis preferably to be understood that the positions of nucleotides/aminoacids may differ in the indicated number than similar neighbouringnucleotides/amino acids, but said neighbouring nucleotides/amino acids,which may be exchanged, deleted, or added, are also comprised by the oneor more corresponding positions.

In addition, for a corresponding position in a lipocalin mutein based ona reference scaffold in accordance with the disclosure, it is preferablyto be understood that the positions of nucleotides/amino acids arestructurally corresponding to the positions elsewhere in a (mutant orwild-type) lipocalin, even if they may differ in the indicated number,as appreciated by the skilled in light of the highly-conserved overallfolding pattern among lipocalins.

The term “albumin” includes all mammal albumins such as human serumalbumin or bovine serum albumin or rat serum albumin.

The term “organic molecule” or “small organic molecule” as used hereinfor the non-natural target denotes an organic molecule comprising atleast two carbon atoms, but preferably not more than 7 or 12 rotatablecarbon bonds, having a molecular weight in the range between 100 and2000 Dalton, preferably between 100 and 1000 Dalton, and optionallyincluding one or two metal atoms.

The word “detect”, “detection”, “detectable” or “detecting” as usedherein is understood both on a quantitative and a qualitative level, aswell as a combination thereof. It thus includes quantitative,semi-quantitative and qualitative measurements of a molecule ofinterest.

A “subject” is a vertebrate, preferably a mammal, more preferably ahuman. The term “mammal” is used herein to refer to any animalclassified as a mammal, including, without limitation, humans, domesticand farm animals, and zoo, sports, or pet animals, such as sheep, dogs,horses, cats, cows, rats, pigs, apes such as cynomolgous monkeys andetc., to name only a few illustrative examples. Preferably, the mammalherein is human.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations.

A “sample” is defined as a biological sample taken from any subject.Biological samples include, but are not limited to, blood, serum, urine,feces, semen, or tissue.

A “fusion polypeptide” as described herein comprises at least twosubunits, wherein one subunit has binding specificity for IL-17A or hasbinding specificity for IL-23p19. Within the fusion polypeptide, thesesubunits may be linked by covalent or non-covalent linkage. Preferably,the fusion polypeptide is a translational fusion between the two or moresubunits. The translational fusion may be generated by geneticallyengineering the coding sequence for one subunit in frame with the codingsequence of a further subunit. Both subunits may be interspersed by anucleotide sequence encoding a linker. However, the subunits of a fusionpolypeptide of the present disclosure may also be linked by a chemicallinker.

A “linker” that may be comprised by a fusion polypeptide of the presentdisclosure links two or more subunits of a fusion polypeptide asdescribed herein. The linkage can be covalent or non-covalent. Apreferred covalent linkage is via a peptide bond, such as a peptide bondbetween amino acids. Accordingly, in a preferred embodiment said linkercomprises of one or more amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids.Preferred linkers are described herein. Other preferred linkers arechemical linkers.

III. DESCRIPTIONS OF FIGURES

FIG. 1: provides a typical measurement of on-rate and off-rate bysurface plasmon resonance for the interaction of the lipocalin mutein ofSEQ ID NO: 1 with human IL-17A. IL-17A was immobilized on a sensor chipusing standard amine chemistry, and the lipocalin mutein of SEQ ID NO: 1was employed as the soluble analyte which was flowed across the chipsurface. The resulting dissociation constant (K_(D)) is 0.8 nM.

FIG. 2: provides a typical measurement of on-rate and off-rate bysurface plasmon resonance for the interaction of the lipocalin mutein ofSEQ ID NO: 1 with human IL-17 A/F. Biotinylated SEQ ID NO: 1 wascaptured on a sensor chip using a dedicated experimental kit, and humanIL-17 A/F was employed as the soluble analyte which was flowed acrossthe chip surface. The resulting dissociation constant (K_(D)) is 100 pM.

FIG. 3: demonstrates that the lipocalin mutein of SEQ ID NO: 1 iscapable of blocking the interaction between hIL-17A and its receptorhIL-17RA with an IC50 of 75 pM. Biotinylated hIL-17A was pre-incubatedwith variable concentrations of the lipocalin mutein of SEQ ID NO: 1 andnon-neutralized hIL-17A was quantified on an ELISA plate withimmobilized soluble hIL-17RA. The negative control SEQ ID NO: 41 had nocompetitive effect. The data were fitted with a single-site bindingmodel.

FIG. 4: shows the crossreactivity profile of the lipocalin mutein of SEQID NO: 1 as measured in a competition ELISA format. Full crossreactivitywith cynomolgus monkey IL-17A and marmoset IL-17A is evident from nearlyidentical IC50 values compared to hIL-17A. The data were fitted with asingle-site binding model.

FIG. 5: illustrates that the lipocalin mutein of SEQ ID NO: 1 is highlyeffective in blocking hIL-17A binding to its receptor hIL-17RA in acell-based assay. The assay is based on hIL-17A-induced secretion ofG-CSF in U87-MG cells. Cells were incubated with a fixed concentrationof hIL-17A and titrated with the lipocalin mutein of SEQ ID NO: 1 or,for comparison, benchmark antibody 1 (heavy chain SEQ ID NO: 53, lightchain SEQ ID NO: 54), benchmark antibody 2 (heavy chain SEQ ID NO: 55,light chain SEQ ID NO: 56) and SEQ ID NO: 2 as a negative control.Plotted is the concentration of G-CSF in arbitrary units as measured byMSD (Meso Scale Discovery®, hereafter “MSD”) against the concentrationof lipocalin muteins or antibody molecules. The resulting average IC50value for the lipocalin mutein of SEQ ID NO: 1 was 0.13 nM (0.17 nM inthe first experiment, 0.10 nM in the repeat experiment), which wassignificantly more potent that benchmark 1, which exhibited an averageIC50=2.33 (2.65/2.01) nM, and in a similar range compared to benchmark2, with an average IC50=0.12 (0.14/0.10) nM. The negative control SEQ IDNO: 2 had no effect on IL-17A-induced G-CSF production of the cells.Binding of SEQ ID NO: 1 or benchmark antibody molecules to IL-17A blocksIL-17A's binding to cell-surface IL-17RA and, thus, prevents inductionof G-CSF secretion. The data were fitted with a single-site bindingmodel, assuming equal G-CSF concentration plateaus for all molecules.

FIG. 6: provides a typical measurement of on-rate and off-rate bysurface plasmon resonance for the lipocalin mutein of SEQ ID NO: 2interacting with human IL-23. The average dissocation constantdetermined in three replicate experiments amounted to K_(D)=0.35±0.20nM.

FIG. 7: provides a typical measurement of on-rate and off-rate bysurface plasmon resonance for the interaction of the lipocalin mutein ofSEQ ID NO: 2 with human IL-23. Biotinylated SEQ ID NO: 2 was captured ona sensor chip using a dedicated experimental kit, and IL-23 was employedas the soluble analyte which was flowed across the chip surface. Theresulting dissociation constant (K_(D)) is 2.9 nM. Note that high,nonphysiological concentrations of NaCl had to be employed to facilitatecarrying out the assay. The result is therefore not representative ofthe affinity of SEQ ID NO: 2 to IL-23 under physiological conditions.The utility of the assay lies in its ability to allow comparisons of theaffinity of SEQ ID NO: 2 and fusion proteins containing this mutein (seeExample 11 and Table 1).

FIG. 8: demonstrates that the lipocalin mutein of SEQ ID NO: 2 iscapable of blocking the interaction between hIL-23 and its receptorhIL-23R with an IC50 of 0.54 nM. Biotinylated hIL-23 was pre-incubatedwith variable concentrations of the lipocalin mutein of SEQ ID NO: 2 andnon-neutralized hIL-23 was quantified on an ELISA plate with immobilizedsoluble hIL-23R. The negative control SEQ ID: 43 has no competitiveeffect. The data were fitted with a single-site binding model.

FIG. 9: shows the crossreactivity profile and specificity of thelipocalin mutein of SEQ ID NO: 2 as measured in a competition ELISAformat. The lipocalin mutein of SEQ ID NO: 2 is fully crossreactive withhuman and mouse IL-23, and displays a somewhat reduced affinity towardscynomolgus monkey and marmoset IL-23. The data were fitted with asingle-site binding model.

FIG. 10: demonstrates that the lipocalin mutein of SEQ ID NO: 2 iscapable of blocking the biological activity of hIL-23 in a cell-basedproliferation assay. In the assay, SEQ ID NO: 2, an IgG isotype negativecontrol and two benchmark antibodies (benchmark 3: heavy chain, SEQ IDNO: 57 and, light chain, SEQ ID NO: 58; benchmark 4: heavy chain, SEQ IDNO: 59 and light chain, SEQ ID NO: 60) were preincubated with hIL-23 andsubsequently added to Ba/F3 cells transfected with hIL-23R andhIL-12Rβ1. The transfected Ba/F3 cells proliferate in response to humanIL-23. The experiment shows that this biological activity is blocked bySEQ ID NO: 2 and the benchmark antibodies 3 and 4 with EC50 values of1.2 nM (1.7/0.7), 3.0 nM (3.1/2.9), 1.2 nM (0.8/1.5), respectively. Thenegative control had no effect on cell proliferation. The data werefitted with a sigmoidal dose-response model.

FIG. 11: provides a graphical overview over the constructs SEQ ID NOs:1-13 characterised in Table 1. SEQ ID NO: 1 (abbreviated in FIG. 11 as“1”) corresponds to the IL-17A-binding lipocalin mutein. SEQ ID NO: 2(abbreviated as “2”) corresponds to an IL-23-binding lipocalin mutein.SEQ ID NO: 14 (abbreviated as “14”) corresponds to the albumin bindingdomain of streptococcal protein G. SEQ ID NO: 15 (abbreviated as “15”)is an engineered, deimmunized version of SEQ ID NO: 14. SEQ ID NO: 16(abbreviated as “16”) corresponds to the Fc-part of a human IgG1antibody.

FIG. 12: demonstrates in an exemplary experiment that a multispecificfusion protein based on the lipocalin muteins disclosed herein iscapable of binding IL-17A, IL-23 and human serum albumin (HSA)simultaneously, without interference by the respective other targetsthat are bound. SEQ ID NO: 9 is a heterodimeric fusion protein of theIL-17A binding lipocalin mutein SEQ ID NO: 1, the IL-23-bindinglipocalin mutein SEQ ID NO: 2 and a human serum albumin binding peptidederived from the albumin binding domain of streptococcal protein G. Inthe surface plasmon resonance experiment shown in FIG. 12, biotinylatedSEQ ID NO: 9 was captured on a sensor chip. To demonstrate simultaneousbinding, dilutions of hIL-17AF, hIL-23 and HSA in buffer wereconsecutively applied to the prepared chip surface. The application ofhIL-17AF, hIL-23 and HSA to immobilized SEQ ID NO: 9 was also performedemploying the single target to obtain the maximum binding levelsobtainable by binding a single target for comparison. The FIG. 12 showsthe measured binding curve and a theoretical binding curve reflectingthe response expected for complete binding of all three targets. Thelatter was obtained by assembling the experimental response of SEQ IDNO: 9 to the individual targets. The measured and the theoretical curveare nearly identical, with the exhibited difference attributable todissociation of the targets in the experimental curve. The data showsthat SEQ ID NO: 9 is capable of simultaneously binding all targetswithout a loss of signal intensity or a change in kinetics compared tobinding a single target only.

FIG. 13: provides typical measurements of on-rate and off-rate bySurface Plasmon Resonance for the lipocalin muteins SEQ ID NO: 45 (FIG.13A) and SEQ ID NO: 46 (FIG. 13B) binding to human IL-23. The resultingdissociation constants (K_(D)) are 0-1 nM (SEQ ID NO: 45) and 0.6 nM(SEQ ID NO: 46), respectively.

FIG. 14: demonstrates that the lipocalin muteins SEQ ID NO: 45 and SEQID NO: 46 are capable of blocking the interaction between hIL-23 and itsreceptor hIL-23R with an IC50 of 0.1 nM (SEQ ID NO: 45) and 1.1 nM (SEQID NO: 46), respectively. Biotinylated hIL-23 was pre-incubated withvariable concentrations of said lipocalin muteins and non-neutralizedhIL-23 was quantified on an ELISA plate with immobilized solublehIL-23R. The data were fitted with a single-site binding model.

FIG. 15: demonstrates that the lipocalin muteins of SEQ ID NO: 45 andSEQ ID NO: 46 are capable of blocking the biological activity of hIL-23in a cell-based proliferation assay. In the assay, SEQ ID NO: 45, SEQ IDNO: 46 and the negative control SEQ ID NO: 43 were preincubated withhIL-23 and subsequently added to Ba/F3 cells transfected with hIL-23Rand hIL-12Rβ1. The transfected Ba/F3 cells proliferate in response tohuman IL-23. The experiment shows that this biological activity isblocked by SEQ ID NO: 45, SEQ ID NO: 46 with IC50 values of 3.7 nM, and5.4 nM, respectively. The negative control SEQ ID NO: 43 had no effecton cell proliferation. The data were fitted with a sigmoidaldose-response model.

FIG. 16: provides a representative experiment in which the specificityof SEQ ID NOs: 63 and 62 and the lipocalin mutein of SEQ ID NO: 1towards the target IL-17A was determined. Biotinylated IL-17A wascaptured on a microtiter plate and the test molecules were titrated.Bound test molecules were detected via an HRP-labeled anti-humanTLc-specific antibody as described in Example 16. The data was fittedwith a 1:1 binding model with EC50 value and the maximum signal as freeparameters, and a slope that was fixed to unity.

FIG. 17: provides a representative experiment in which the specificityof SEQ ID NOs: 64 and 62 and the lipocalin mutein of SEQ ID NO: 2towards the target IL-23 was determined. Biotinylated IL-23 was capturedon a microtiter plate, and the test molecules were titrated. Bound testmolecules were detected via an HRP-labeled anti-human NGAL-specificantibody as described in Example 17. The data was fitted with a 1:1binding model with EC50 value and the maximum signal as free parameters,and a slope that was fixed to unity.

FIG. 18: provides a representative experiment in which the specificityof the polypeptide fusion of SEQ ID NOs: 63 and 62 and the polypeptidefusion of SEQ ID NOs: 64 and 62, as well as that of the antibody of SEQID NOs: 61 and 62 towards the target TNF-α was determined. TNF-α wascoated on a microtiter plate, and the test molecules were titrated.Bound test molecules were detected via an HRP-labeled anti-human IgGFc-specific antibody as described in Example 18. The data was fittedwith a 1:1 binding model with EC50 value and the maximum signal as freeparameters, and a slope that was fixed to unity.

FIG. 19: provides a representative experiment in which the ability ofthe polypeptide fusion of SEQ ID NOs: 63 and 62 and the polypeptidefusion of SEQ ID NOs: 64 and 62 to simultaneously bind bothtargets—TNF-α and IL-17A, TNF-α and IL-23, respectively—was determined.Recombinant TNF-α was coated on a microtiter plate, followed by atitration of the polypeptide fusions. Subsequently, a constantconcentration of either biotinylated IL-17A or IL-23 was added, whichwas detected via HRP-labeled extravidin as described in Example 19. Thedata was fitted with a 1:1 binding model with EC50 value and the maximumsignal as free parameters, and a slope that was fixed to unity.

IV. DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure contributes to the state of art a polypeptide orprotein having binding specificity for IL-17A and/or IL-23p19, whereinthe polypeptide comprises a lipocalin mutein that binds with at least adetectable affinity to IL-17A or IL-23p19.

In some embodiments, the polypeptide is a lipocalin mutein that iscapable of binding IL-17A with at least a detectable affinity. In someembodiments, the polypeptide is a lipocalin mutein that is capable ofbinding IL-23p19 with at least a detectable affinity. The presentdisclosure also relates to use of both polypeptides, for the binding ofIL-17A and IL-23p19 in a subject.

In some aspects, the polypeptide is a fusion protein comprising at leasttwo subunits, wherein one subunit has binding specificity for IL-17A andanother subunit has binding specificity for IL-23p19. In some furtherembodiments, the fusion protein may further comprise a subunit, whereinthe subunit has binding specificity for IL-23p19 or IL-17A. In somestill further embodiments, the fusion protein may comprise one subunitspecific for IL-17A, one subunit specific for IL-23p19, and one subunitcontaining a bacterial albumin binding domain (ABD).

In some other aspects, a polypeptide of the disclosure may also be afusion protein comprising at least two subunits specific for IL-17A, ora fusion protein comprising at least two subunits specific for IL-23p19.

In some embodiments, the subunit of the fusion protein having bindingspecificity for IL-17A comprises a lipocalin mutein specific for IL-17Aof the disclosure. In some embodiments, the subunit of the fusionprotein having binding specificity for IL-23p19 comprises an antibodythat binds to IL-23p19. In some other embodiments, the subunit of thefusion protein having binding specificity for IL-23p19 comprises alipocalin mutein specific for IL-23p19 of the disclosure. In someembodiments, the subunit of the fusion protein having bindingspecificity for IL-17A comprises an antibody that binds to IL-17A.

A polypeptide or protein of the disclosure can be a mutein of alipocalin, preferably a lipocalin selected from the group consisting ofretinol-binding protein (RBP), bilin-binding protein (BBP),apolipoprotein D (APO D), neutrophil gelatinase associated lipocalin(NGAL), tear lipocalin (TLPC or Tlc), α₂-microglobulin-related protein(A2m), 24p3/uterocalin (24p3), von Ebners gland protein 1 (VEGP 1), vonEbners gland protein 2 (VEGP 2), and Major allergen Can f1 precursor(ALL-1).

As used herein, a “lipocalin” is defined as a monomeric protein ofapproximately 18-20 kDA in weight, having a cylindrical β-pleated sheetsupersecondary structural region comprising a plurality of (preferablyeight) β-strands connected pair-wise by a plurality of (preferably four)loops at one end to define thereby a binding pocket. It is the diversityof the loops in the otherwise rigid lipocalin scaffold that gives riseto a variety of different binding modes among the lipocalin familymembers, each capable of accommodating targets of different size, shape,and chemical character (reviewed, e.g., in Flower, D. R. (1996), supra;Flower, D. R. et al. (2000), supra, or Skerra, A. (2000) Biochim.Biophys. Acta 1482, 337-350). Indeed, the lipocalin family of proteinshave naturally evolved to bind a wide spectrum of ligands, sharingunusually low levels of overall sequence conservation (often withsequence identities of less than 20%) yet retaining a highly conservedoverall folding pattern. The correspondence between positions in variouslipocalins is well known to one of skill in the art. See, for example,U.S. Pat. No. 7,250,297.

As noted above, a lipocalin is a polypeptide defined by itssupersecondary structure, namely cylindrical β-pleated sheetsupersecondary structural region comprising eight β-strands connectedpair-wise by four loops at one end to define thereby a binding pocket.The present disclosure is not limited to lipocalin muteins specificallydisclosed herein. In this regard, the disclosure relates to a lipocalinmutein having a cylindrical 1-pleated sheet supersecondary structuralregion comprising eight β-strands connected pair-wise by four loops atone end to define thereby a binding pocket, wherein at least one aminoacid of each of at least three of said four loops has been mutated andwherein said lipocalin is effective to bind IL-17A or IL-23p19 withdetectable affinity.

In one particular embodiment, a lipocalin mutein disclosed herein is amutein of human tear lipocalin (TLPC or Tlc), also termed lipocalin-1,tear pre-albumin or von Ebner gland protein. The term “human tearlipocalin” or “Tlc” or “lipocalin-1” as used herein refers to the maturehuman tear lipocalin with the SWISS-PROT/UniProt Data Bank AccessionNumber P31025 (Isoform 1). The amino acid sequence shown inSWISS-PROT/UniProt Data Bank Accession Number P31025 may be used as apreferred “reference sequence”, more preferably the amino acid sequenceshown in SEQ ID NO: 41 is used as reference sequence.

The present disclosure also encompasses Tlc muteins as defined above, inwhich the first four N-terminal amino acid residues of the sequence ofmature human tear lipocalin (His-His-Leu-Leu; positions 1-4) and/or thelast two C-terminal amino acid residues (Ser-Asp; positions 157-158) ofthe linear polypeptide sequence of the mature human tear lipocalin(SWISS-PROT Data Bank Accession Number P31025) have been deleted (SEQ IDNOs: 2-5). In addition, the present disclosure encompasses Tlc muteinsas defined above, in which one GH loop amino acid residue (Lys)corresponding to sequence position 108 of the linear polypeptidesequence of the mature human tear lipocalin has been deleted (SEQ ID NO:1 and SEQ ID NO: 43). Another possible mutation of the wild typepolypeptide sequence of the mature human tear lipocalin is to change theamino acid sequence at sequence positions 5 to 7 (Ala Ser Asp) to GlyGly Asp as described in PCT application WO 2005/019256, which isincorporated by reference its entirety herein.

A Tlc mutein according to the disclosure may further include an aminoacid substitution Arg 111→Pro. A Tlc mutein according to the disclosuremay also include a substitution Lys 114→Trp. It may also comprise asubstitution Cys 101→Ser or Cys 101→Thr. In some preferred embodiments,a Tlc mutein according to the disclosure may also comprise asubstitution Cys 153→Ser.

Modifications of the amino acid sequence include directed mutagenesis ofsingle amino acid positions in order to simplify sub-cloning of themutated lipocalin gene or its parts by incorporating cleavage sites forcertain restriction enzymes. In addition, these mutations can also beincorporated to further improve the affinity of a Tlc mutein for IL-17Aor IL-23p19. Furthermore, mutations can be introduced in order tomodulate certain characteristics of the mutein such as to improvefolding stability, serum stability, protein resistance or watersolubility or to reduce aggregation tendency, if necessary. For example,naturally occurring cysteine residues may be mutated to other aminoacids to prevent disulphide bridge formation. Exemplary possibilities ofsuch a mutation to introduce a cysteine residue into the amino acidsequence of a Tlc mutein include the substitutions Thr 40→Cys, Glu73→Cys, Arg 90→Cys, Asp 95→Cys, and Glu 131→Cys. The generated thiolmoiety at the side of any of the amino acid positions 40, 73, 90, 95and/or 131 may be used to PEGylate or HESylate the mutein, for example,in order to increase the serum half-life of a respective Tlc mutein.

In another particular embodiment, a lipocalin mutein disclosed herein isa mutein of human lipocalin 2. The term “human lipocalin 2” or “humanLcn 2” or “human NGAL” as used herein refers to the mature humanneutrophil gelatinase-associated lipocalin (NGAL) with theSWISS-PROT/UniProt Data Bank Accession Number P80188. A human lipocalin2 mutein of the disclosure may also be designated herein as “an hNGALmutein”. The amino acid sequence shown in SWISS-PROT/UniProt Data BankAccession Number P80188 may be used as a preferred “reference sequence”,more preferably the amino acid sequence shown in SEQ ID NO: 43 is usedas reference sequence.

In some embodiments, a lipocalin mutein binding IL-17A or IL-23p19 withdetectable affinity may include at least one amino acid substitution ofa native cysteine residue by another amino acid, for example, a serineresidue. In some other embodiments, a lipocalin mutein binding IL-17A orIL-23p19 with detectable affinity may include one or more non-nativecysteine residues substituting one or more amino acids of a wild typelipocalin. In a further particular embodiment, a lipocalin muteinaccording to the disclosure includes at least two amino acidsubstitutions of a native amino acid by a cysteine residue, hereby toform one or more cysteine bridges. In some embodiments, said cysteinebridge may connect at least two loop regions. The definition of theseregions is used herein in accordance with Flower (Flower, 1996, supra,Flower, et al., 2000, supra) and Breustedt et al. (2005, supra).

Proteins of the disclosure, which are directed against or specific forIL-17A or IL-23p19, include any number of specific-binding proteinmuteins that are based on a defined protein scaffold. Preferably, thenumber of nucleotides or amino acids, respectively, that is exchanged,deleted or inserted is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or more such as 25, 30, 35, 40, 45 or 50, with 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 being preferred and 9, 10 or 11 beingeven more preferred. However, it is preferred that a lipocalin mutein ofthe disclosure is still capable of binding IL-17A or IL-23p19, inparticular human IL-17A or human IL-23p19.

In one aspect, the present disclosure includes various lipocalin muteinsthat bind IL-17A or IL-23p19 with at least detectable affinity. In thissense, IL-17A or IL-23p19 can be regarded a non-natural ligand of thereference wild type lipocalin, where “non-natural ligand” refers to acompound that does not bind to wildtype lipocalins under physiologicalconditions. By engineering wildtype lipocalins with one or moremutations at certain sequence positions, the present inventors havedemonstrated that high affinity and high specificity for the non-naturalligand, e.g. IL-17A or IL-23p19, is possible. In some embodiments, at 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or even more nucleotide triplet(s)encoding certain sequence positions on wildtype lipocalins, a randommutagenesis may be carried out through substitution at these positionsby a subset of nucleotide triplets.

Further, the lipocalin muteins of the disclosure may have a mutatedamino acid residue at any one or more, including at least at any one,two, three, four, five, six, seven, eight, nine, ten, eleven or twelve,of the sequence positions corresponding to certain sequence positions ofthe linear polypeptide sequence of the reference lipocalin.

A protein of the disclosure may include the wild type (natural) aminoacid sequence of the “parental” protein scaffold (such as a lipocalin)outside the mutated amino acid sequence positions. In some embodiments,a lipocalin mutein according to the disclosure may also carry one ormore amino acid mutations at a sequence position/positions as long assuch a mutation does, at least essentially not hamper or not interferewith the binding activity and the folding of the mutein. Such mutationscan be accomplished very easily on DNA level using established standardmethods (Sambrook, J. et al. (2001) Molecular Cloning: A LaboratoryManual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.). Illustrative examples of alterations of the amino acidsequence are insertions or deletions as well as amino acidsubstitutions. Such substitutions may be conservative, i.e. an aminoacid residue is replaced with an amino acid residue of chemicallysimilar properties, in particular with regard to polarity as well assize. Examples of conservative substitutions are the replacements amongthe members of the following groups: 1) alanine, serine, and threonine;2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4)arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and6) phenylalanine, tyrosine, and tryptophan. On the other hand, it isalso possible to introduce non-conservative alterations in the aminoacid sequence. In addition, instead of replacing single amino acidresidues, it is also possible to either insert or delete one or morecontinuous amino acids of the primary structure of the human tearlipocalin as long as these deletions or insertion result in a stablefolded/functional mutein (for example, Tlc muteins with truncated N- andC-terminus). In such mutein, for instance, one or more amino acidresidues are added or deleted at the N- or C-terminus of thepolypeptide. Generally such a mutein may have about at least 70%,including at least about 80%, such as at least about 85% amino acidsequence identity, with the amino acid sequence of the mature human tearlipocalin. As an illustrative example, the present disclosure alsoencompasses Tlc muteins as defined above, in which the first fourN-terminal amino acid residues of the sequence of mature human tearlipocalin (His-His-Leu-Leu; positions 1-4) and/or the last twoC-terminal amino acid residues (Ser-Asp; positions 157-158) of thelinear polypeptide sequence of the mature human tear lipocalin have beendeleted (SEQ ID NO: 1 and SEQ ID NO: 43).

The amino acid sequence of a lipocalin mutein disclosed herein has ahigh sequence identity to the reference lipocalin when compared tosequence identities with other lipocalins. In this general context, theamino acid sequence of a lipocalin mutein of the disclosure is at leastsubstantially similar to the amino acid sequence of the referencelipocalin, with the proviso that possibly there are gaps (as definedbelow) in an alignment that are the result of additions or deletions ofamino acids. A respective sequence of a lipocalin mutein of thedisclosure, being substantially similar to the sequences of thereference lipocalin, has, in some embodiments, at least 70% identity orsequence homology, at least 75% identity or sequence homology, at least80% identity or sequence homology, at least 82% identity or sequencehomology, at least 85% identity or sequence homology, at least 87%identity or sequence homology, or at least 90% identity or sequencehomology including at least 95% identity or sequence homology, to thesequence of the reference lipocalin, with the proviso that the alteredposition or sequence is retained and that one or more gaps are possible.

As used herein, a lipocalin mutein of the disclosure “specificallybinds” a target (for example, IL-17A or IL-23p19) if it is able todiscriminate between that target and one or more reference targets,since binding specificity is not an absolute, but a relative property.“Specific binding” can be determined, for example, in accordance withWestern blots, ELISA-, RIA-, ECL-, IRMA-tests, FACS, IHC and peptidescans.

In one embodiment, the lipocalin muteins of the disclosure are fused atits N-terminus and/or its C-terminus to a fusion partner which is aprotein domain that extends the serum half-life of the mutein. Infurther particular embodiments, the protein domain is a Fc part of animmunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of animmunoglobulin, an albumin binding domain, an albumin binding peptide,or an albumin binding protein.

In another embodiment, the lipocalin muteins of the disclosure areconjugated to a compound that extends the serum half-life of the mutein.More preferably, the mutein is conjugated to a compound selected fromthe group consisting of a polyalkylene glycol molecule, ahydroethylstarch, an Fc part of an immunoglobulin, a CH3 domain of animmoglobulin, a CH4 domain of an immunoglobulin, an albumin bindingdomain, an albumin binding peptide, and an albumin binding protein.

In yet another embodiment, the current disclosure relates to a nucleicacid molecule comprising a nucleotide sequence encoding a lipocalinmutein disclosed herein. The disclosure encompasses a host cellcontaining said nucleic acid molecule.

A Tlc mutein according to the present disclosure can be obtained bymeans of mutagenesis of a naturally occurring form of human tearlipocalin. An hNGAL mutein according to the present disclosure can beobtained by means of mutagenesis of a naturally occurring form of humanlipocalin 2. In some embodiments of the mutagenesis, a substitution (orreplacement) is a conservative substitution. Nevertheless, anysubstitution—including non-conservative substitution or one or more fromthe exemplary substitutions below—is envisaged as long as the lipocalinmutein retains its capability to bind to IL-17A or IL-23p19, and/or ithas an identity to the then substituted sequence in that it is at least60%, such as at least 65%, at least 70%, at least 75%, at least 80%, atleast 85% or higher identity to the amino acid sequence of the maturehuman tear lipocalin or the mature human lipocalin 2, respectively.

A. Lipocalin Muteins with Binding-Affinity for Interleukin-17A (IL-17A,Synonymous with IL-17)

In one aspect, the present disclosure provides human lipocalin muteinsthat bind human IL-17A (same as “IL-17”) and useful applicationstherefor. Binding proteins described herein may bind human IL-17Ahomodimer (same as “IL-17 A”) and/or heterodimers of human IL-17A andthe human IL-17F homolog (same as “IL-17 A/F”). The disclosure alsoprovides methods of making IL-17A binding proteins described herein aswell as compositions comprising such proteins. IL-17A binding proteinsof the disclosure as well as compositions thereof may be used in methodsof detecting IL-17A (including IL-17 A/A and IL-17 A/F) in a sample orin methods of binding IL-17A (including IL-17 A/A and IL-17 A/F) in asubject. No such human lipocalin muteins having these features attendantto the uses provided by present disclosure have been previouslydescribed.

One embodiment of the current disclosure relates to a lipocalin muteinthat is capable of binding Interleukin-17A (IL-17A) with an affinitymeasured by a K_(D) of about 1 nM or lower, such as 0.8 nM, whenmeasured in an assay essentially described in Example 1.

In some other embodiments, the lipocalin mutein is capable of inhibitingthe binding of IL-17A to its receptor IL-17RA with an IC50 value ofabout 100 pM or lower, such as 75 pM, in a competition ELISA formatessentially described in Example 3.

In some particular embodiments, the IL-17A-binding lipocalin mutein iscrossreactive with human IL-17A, cynomolgus IL-17A and marmoset monkeyIL-17A.

In some still further embodiments, a lipocalin mutein of the disclosureis capable of blocking IL-17A binding to its receptor IL-17RA. In somefurther embodiments, the lipocalin mutein has an average EC50 value atleast as good as (i.e. where in difference is less than 0.1 nM) orsuperior to the EC50 value of a benchmark antibody, when said lipocalinmutein and the benchmark antibody are measured in an assay essentiallyas described in Example 5. In some embodiments, the benchmark antibodyis a polypeptide comprising (i) SEQ ID NO: 53 or 55 as the first subunitand (ii) SEQ ID NO: 54 or 56 as the second subunit. The lipocalin muteinmay have an average IC50 value of about 0.13 nM or even lower in theassay when at the same time the benchmark antibody has an EC50 value ofabout 2.33 nM or lower in the assay, such as about 0.12 nM.

In some other embodiments, an IL-17A-binding lipocalin mutein of thecurrent disclosure is capable of binding IL-17A with a higher affinitythan the lipocalin mutein of SEQ ID NO: 42, measured by a lower K_(D) ofthe first said lipocalin mutein than the K_(D) of the lipocalin muteinof SEQ ID NO: 42, for example, in an assay essentially described inExample 1. In some further embodiments, an IL-17A-binding lipocalinmutein of the current disclosure is capable of inhibiting the binding ofIL-17A to its receptor IL-17RA with a lower EC50 value than that of thelipocalin mutein of SEQ ID NO: 42, for example, when measured in anassay essentially described in Example 5.

1. Exemplary Lipocalin Muteins with Binding-Affinity for Interleukin-17A(IL-17A)

In one aspect, the present disclosure relates to novel, specific-bindinghuman tear lipocalin muteins directed against or specific forInterleukin-17A (IL-17A). Human tear lipocalin muteins disclosed hereinmay be used for therapeutic and/or diagnostic purposes. A human tearlipocalin mutein of the disclosure may also be designated herein as “aTlc mutein”. As used herein, a Tlc mutein of the disclosure“specifically binds” a target (e.g. here, IL-17A) if it is able todiscriminate between that target and one or more reference targets,since binding specificity is not an absolute, but a relative property.“Specific binding” can be determined, for example, in accordance withWestern blots, ELISA-, RIA-, ECL-, IRMA-tests, FACS, IHC and peptidescans.

In this regard, the disclosure provides one or more Tlc muteins that arecapable of binding Interleukin-17A (IL-17A) with an affinity measured bya KD of about 10 nM, about 1 nM, about 0.1 nM or lower. More preferably,the Tlc muteins can have an affinity measured by a KD of about 1 nM, 0.8nM, 0.6 nM, 100 pM or lower.

In some particular embodiments, such Tlc mutein includes a mutated aminoacid residue at one or more positions corresponding to positions 26-33,56, 58, 60-61, 64, 92, 101, 104-106, 108, 111, 114 and 153 of the linearpolypeptide sequence of the mature human tear lipocalin (SWISS-PROT DataBank Accession Number P31025; SEQ ID NO: 41).

In further particular embodiments, such Tlc mutein may further include amutated amino acid residue at one or more positions corresponding topositions 101, 111, 114 and 153 of the linear polypeptide sequence ofthe mature human tear lipocalin (SEQ ID NO: 41).

In some further embodiments, the Tlc mutein may contain at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, oreven more, mutated amino acid residues at one or more sequence positionscorresponding to sequence positions 26, 27, 28, 29, 30, 31, 32, 33, 56,58, 60, 61, 64, 92, 101, 104, 105, 106, 108, 111, 114 and 153 ofthelinear polypeptide sequence of the mature human tear lipocalin (SEQ IDNO: 41).

In some still further embodiments, the disclosure relates to apolypeptide, wherein said polypeptide is a Tlc mutein, in comparisonwith the linear polypeptide sequence of the mature human tear lipocalin,comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or even more,mutated amino acid residues at the sequence positions 26-33, 56, 58,60-61, 64, 92, 101, 104-106, 108, 111, 114 and 153 and wherein saidpolypeptide binds IL-17A, in particular human IL-17A.

In some embodiments, a lipocalin mutein according to the disclosure mayinclude at least one amino acid substitution of a native cysteineresidue by e.g. a serine residue. In some embodiments, a Tlc muteinaccording to the disclosure includes an amino acid substitution of anative cysteine residue at positions 61 and/or 153 by a serine residue.In this context it is noted that it has been found that removal of thestructural disulfide bond (on the level of a respective naïve nucleicacid library) of wild type tear lipocalin that is formed by the cysteineresidues 61 and 153 (cf. Breustedt, et al., 2005, supra) may providetear lipocalin muteins that are not only stably folded but are also ableto bind a given non-natural ligand with high affinity. Without wishingto be bound by theory, it is also believed that the elimination of thestructural disulde bond provides the further advantage of allowing forthe (spontaneous) generation or deliberate introduction of non-naturalartificial disulfide bonds into muteins of the disclosure, therebyincreasing the stability of the muteins. For example, in someembodiments, a Tlc mutein according to the disclosure includes an aminoacid substitution of a native cysteine residue at position 101 by aserine residue. Further, in some embodiments, a mutein according to thedisclosure includes an amino acid substitution of a native arginineresidue at positions 111 by a proline residue. In some embodiments amutein according to the disclosure includes an amino acid substitutionof a native lysine residue at positions 114 by a tryptophan residue.

A Tlc mutein according to the disclosure may further include, withrespect to the amino acid sequence of the mature human tear lipocalin(SWISS-PROT Data Bank Accession Number P31025), one or more, includingat least two, at least three, at least four, at least five, at leastsix, at least seven, at least eight, at least nine, at least ten, atleast eleven, at least twelve, at least thirteen or at least fourteenamino acid substitutions of native amino acid residues by cysteineresidues at any of positions 26-33, 56, 58, 60-61, 64, 92, 101, 104-106,108, 111, 114 and 153 of the mature human tear lipocalin.

In some embodiments, a mutein according to the disclosure includes anamino acid substitution of a native amino acid by a cysteine residue atpositions 28 or 105 with respect to the amino acid sequence of maturehuman tear lipocalin. In some embodiments a mutein according to thedisclosure includes an amino acid substitution of a native amino acid bya cysteine residue at positions 28 or 105 with respect to the amino acidsequence of mature human tear lipocalin. In a further particularembodiment, a mutein according to the disclosure includes an amino acidsubstitution of a native amino acid by two cysteine residues atpositions 28 and 105 with respect to the amino acid sequence of maturehuman tear lipocalin.

In some embodiments, a Tlc mutein according to the disclosure includes asubstituted amino acid of at least one or of both of the cysteineresidues occurring at each of the sequences positions 61 and 153 byanother amino acid and the mutation of at least three amino acid residueat any one of the sequence positions 26-33, 56, 58, 60-61, 64, 92, 101,104-106, 108, 111, 114 and 153 of the linear polypeptide sequence of themature human tear lipocalin (SWISS-PROT Data Bank Accession NumberP31025). The positions 26-34 are included in the AB loop, the position55 is located at the very end of a beta-sheet and following positions56-58 as well as 60-61 and 64 are included in the CD loop. The positions104-108 are included in the GH loop in the binding site at the open endof the β-barrel structure of the mature human tear lipocalin. Thedefinition of these regions is used herein in accordance with Flower(Flower, 1996, supra, Flower, et al., 2000, supra) and Breustedt et al.(2005, supra). In some embodiments, the Tlc mutein according to thedisclosure includes the amino acid substitutions Cys 61→Ala, Phe, Lys,Arg, Thr, Asn, Gly, Gln, Asp, Asn, Leu, Tyr, Met, Ser, Pro or Trp andCys 153→Ser or Ala. Such a substitution has proven useful to prevent theformation of the naturally occurring disulphide bridge linking Cys 61and Cys 153, and thus to facilitate handling of the mutein. However,tear lipocalin muteins that binds IL-17A and that have the disulphidebridge formed between Cys 61 and Cys 153 are also part of the presentdisclosure.

In some embodiments, an IL-17A-binding Tlc mutein according to thedisclosure includes, at any one or more of the sequence positions 26-33,56, 58, 60-61, 64, 92, 101, 104-106, 108, 111, 114 and 153 of the linearpolypeptide sequence of the mature human tear lipocalin (SEQ ID NO: 41),one or more of the following mutated amino acid residues: Arg 26→Phe;Glu 27→Trpl; Phe 28→Cys; Pro 29→Ser; Glu 30→Gly; Met 31→Ile; Asn 32→His;Leu 33→Glu; Leu 56→Asp; Ser 58→Glu; Arg 60→Phe; Cys 61→Leu; Val 64→Phe;His 92→Arg; Cys101→Ser; Glu 104-Asp; Leu 105→Cys; His 106→Pro; deletionof Lys 108; Arg 111→Pro; Lys 114→Trp; and Cys 153→Ser. In someembodiments, a Tlc mutein of the disclosure includes two or more, suchas 3, 4, 5, 6, 7, 8 or all mutated amino acid residues at these sequencepositions of the mature human tear lipocalin.

In further particular embodiments, a Tlc mutein of the disclosurecomprises an amino acid sequence as set forth in any one of SEQ ID NOs:1 or a fragment or variant thereof.

In further particular embodiments, a Tlc mutein of the disclosure has atleast 75%, at least 80%, at least 85% or higher identity to an aminoacid sequence selected from the group consisting of SEQ ID NOs: 1.

The disclosure also includes structural homologues of a Tlc muteinhaving an amino acid sequence selected from the group consisting of SEQID NOs: 1, which structural homologues have an amino acid sequencehomology or sequence identity of more than about 60%, preferably morethan 65%, more than 70%, more than 75%, more than 80%, more than 85%,more than 90%, more than 92% and most preferably more than 95% inrelation to said Tlc mutein.

In some particular embodiments, the present disclosure provides alipocalin mutein that binds IL-17A with an affinity measured by a KD ofabout 1 nM or lower, wherein the lipocalin mutein has at least 90% orhigher, such as 95%, identity to the amino acid sequence of SEQ ID NO:1.

2. Applications of Lipocalin Muteins with Binding-Affinity forInterleukin-17A (IL-17A)

IL-17A is a pro-inflammatory cytokine produced by a subset of memory Tcells (called Th17) that has been implicated in the pathogenesis of manydisorders, e.g. multiple sclerosis (MS) (Hellings, P. W. et al., Am. J.Resp. Cell Mol. Biol. 28 (2003) 42-50; Matusevicius, D. et al., MultipleSclerosis 5 (1999) 101-104), rheumatoid arthritis (RA) (Ziolkovvska, M.et al., J. Immunol. 164 (2000) 2832-38; Kotake, S. et al., J. Clin.Invest. 103 (1999) 1345-52; Hellings, P. W. et al., Am. J. Resp. CellMol. Biol. 28 (2003) 42-50). IL-17A plays a role in the induction ofother inflammatory cytokines, chemokines and adhesion molecules(Komiyama, Y. et al., J. Immunol. 177 (2006) 566-573), psoriasis,Crohn's disease, chronic obstructive pulmonary disease (COPD), asthma,and transplant rejection.

IL-17A is involved in the induction of proinflammatory responses andinduces or mediates expression of a variety of other cytokines, factors,and mediators including tissue necrosis factor-alpha (TNF-α), IL-6,IL-8, IL-1p, granulocyte colony-stimulating factor (G-CSF),prostaglandin E2 (PGE2), IL-10, IL-12, IL-IR antagonist, leukemiainhibitory factor, and stromelysin (Yao et al., J. Immunol, 155(12):5483-5486 (1995); Fossiez et al., J. Exp. Med., 183(6): 2593-2603(1996); Jovanovic et al., J. Immunol, 160: 3513-3521 (1998); Teunissenet al., J. Investig. Dermatol, 111: 645-649 (1998); Chabaud et al., J.Immunol, 161: 409-414 (1998)). IL-17A also induces nitric oxide inchondrocytes and in human osteoarthritis explants (Shalom-Barak et al.,J. Biol Chem., 273: 27467-27473 (1998); Attur et al., Arthritis Rheum.,40: 1050-1053 (1997)). Through its role in T cell mediated autoimmunity,IL-17A induces the release of cytokines, chemokines, and growth factors(as noted above), is an important local orchestrator of neutrophilaccumulation, and plays a role in cartilage and bone destruction. Thereis growing evidence that targeting IL-17A signaling might prove usefulin a variety of autoimmune diseases including rheumatoid arthritis (RA),psoriasis, Crohn's disease, multiple sclerosis (MS), psoriatric disease,asthma, and lupus (SLE) (see, e.g., Aggarwal et al., J. Leukoc. Biol,71(1): 1-8 (2002); Lubberts et al., “Treatment with a neutralizinganti-murine interleukin-17 antibody after the onset of collagen-inducedarthritis reduces joint inflammation, cartilage destruction, and boneerosion,” Arthritis Rheum., 50: 650-659 (2004)).

In addition, it is known in the art that inflammatory andimmunoregulatory processes are implicated in the pathogenesis of variousforms of cardiovascular disease (Biasucci, L., et al., Circulation 1999,99:855-860; Albert, C, et al, Circulation 2002, 105:2595-9; Buffon, A.,et al, NEJM 2002, 347:55-7; Nakajima, T., et al., Circulation 2002,105:570-5). Recent studies have established a basis for treatingcardiovascular disease by reducing inflammatory and immunoregulatoryresponses of the disease (Blankenberg, S., et al., Circulation 2002,106:24-30; Mallat, Z., et al, Circulation 2001, 104:1598-603; Mallat,Z., et al, Circ Res. 2001, 89:E41-5). Cardiovascular disease encompassesa number of disorders that affect the muscle and/or blood vessels of theheart, peripheral blood vessels, muscles and various organs.

Numerous possible applications for the Tlc muteins of the disclosure,therefore, exist in medicine. In one further aspect, the disclosurerelates to the use of a Tlc mutein disclosed for detecting IL-17A(including IL-17 A/A and IL-17 A/F) in a sample as well as a respectivemethod of diagnosis.

The present disclosure also involves the use of one or more Tlc muteinsas described for complex formation with IL-17A.

Therefore, in another aspect of the disclosure, the disclosed muteinsare used for the detection of IL-17A. Such use may include the steps ofcontacting one or more said muteins, under suitable conditions, with asample suspected of containing IL-17A, thereby allowing formation of acomplex between the muteins and IL-17A, and detecting the complex by asuitable signal.

The detectable signal can be caused by a label, as explained above, orby a change of physical properties due to the binding, i.e. the complexformation, itself. One example is plasmon surface resonance, the valueof which is changed during binding of binding partners from which one isimmobilized on a surface such as a gold foil.

The muteins disclosed herein may also be used for the separation ofIL-17A. Such use may include the steps of contacting one or more saidmuteins, under suitable conditions, with a sample supposed to containIL-17A, thereby allowing formation of a complex between the muteins andIL-17A, and separating the complex from the sample.

In the use of the disclosed muteins for the detection of IL-17A as wellas the separation of IL-17A, the muteins and/or IL-17A or a domain orfragment thereof may be immobilized on a suitable solid phase.

In still another aspect, the present disclosure features a diagnostic oranalytical kit comprising a Tlc mutein according to the disclosure.

In addition to their use in diagnostics, in yet another aspect, thedisclosure encompasses the use of a mutein of the disclosure or acomposition comprising such mutein for the binding of IL-17A in asubject and/or inhibiting the binding of IL-17A to its receptor in asubject.

In still another aspect, the present disclosure features a method ofbinding IL-17A in a subject, comprising administering to said subject aneffective amount of one or more lipocalin muteins of the disclosure orof one or more compositions comprising such muteins.

In still another aspect, the present disclosure involves a method forinhibiting the binding of IL-17A to its receptor in a subject,comprising administering to said subject an effective amount of one ormore lipocalin muteins of the disclosure or of one or more compositionscomprising such muteins.

In the context of the present disclosure, the disclosed lipocalinmuteins with binding-affinity for IL-17A can bind to IL-17A that existsas a homodimer, but such muteins can also bind to IL-17A that exists asa heterodimer complexed with the homolog IL-17F to form heterodimericIL-17 A/F. In one preferred embodiment, one lipocalin mutein of thedisclosure may bind with a detectable affinity to IL-17A in complex withIL-17F.

B. Lipocalin Muteins with Binding-Affinity for Interleukin-23p19(IL-23p19)

In addition, the present disclosure fulfills the need for alternativeinhibitors of IL-23p19 by providing human lipocalin muteins that bindhuman IL-23p19 and useful applications therefor. Accordingly, thedisclosure also provides methods of making and using theIL-23p19-binding proteins described herein as well as compositions thatmay be used in methods of detecting IL-23p19 in a sample or in methodsof binding of IL-23p19 in a subject. No such human lipocalin muteinshaving these features attendant to the uses provided by presentdisclosure have been previously described.

One embodiment of the current disclosure relates to a lipocalin muteinthat is capable of binding Interleukin-23p19 (IL-23p19), with anaffinity measured by a K_(D) of about 1 nM or lower, such as about 0.6nM, when measured in an assay essentially described in Example 6 orExample 13.

In some other embodiments, the lipocalin mutein is capable of inhibitingthe binding of IL-23 to its receptor IL-23R with an IC50 value of about0.55 nM or lower in a competition ELISA format essentially described inExample 8 or Example 14.

In some particular embodiments, lipocalin mutein is crossreactive withboth human IL-23 and mouse IL-23.

In some still further embodiments, a lipocalin mutein of the disclosureis capable of inhibiting the binding of IL-23 to its receptor IL-23R. Insome further embodiments, the lipocalin mutein has an average EC50 valueat least as good as (i.e. where the difference is less than 1.0 nM) orsuperior to the average EC50 value of of a benchmark antibody, when saidlipocalin mutein and the benchmark antibody are measured in an assayessentially as described in Example 10 or Example 15. In someembodiments, the benchmark antibody is a polypeptide comprising (i) SEQID NO: 57 or 59 as the first subunit and (ii) SEQ ID NO: 58 or 60 as thesecond subunit. The lipocalin mutein may have an average EC50 value ofabout 1.2 nM or even lower in the assay when at the same time thebenchmark antibody has an EC50 value of about 3 nM or lower in theassay, such as about 1.2 nM.

In some other embodiments, an IL-23p19-binding lipocalin mutein of thedisclosure is more biophyscially stable than the lipocalin mutein of SEQID NO: 44.

1. Exemplary Lipocalin Muteins with Binding-Affinity forInterleukin-23p19 (IL-23p19)

In one aspect, the present disclosure relates to novel, specific-bindinghuman lipocalin 2 (Lcn2 or NGAL) muteins directed against or specificfor Interleukin-23p19 (IL-23p19). Human lipocalin 2 muteins disclosedherein may be used for therapeutic and/or diagnostic purposes. A humanlipocalin 2 mutein of the disclosure may also be designated herein as “aNGAL mutein”. As used herein, a Tlc mutein of the disclosure“specifically binds” a target (here, IL-23p19) if it is able todiscriminate between that target and one or more reference targets,since binding specificity is not an absolute, but a relative property.“Specific binding” can be determined, for example, in accordance withWestern blots, ELISA-, RIA-, ECL-, IRMA-tests, FACS, IHC and peptidescans.

In this regard, the disclosure provides one or more NGAL muteins thatare capable of binding Interleukin-23p19 (IL-23p19) with an affinitymeasured by a KD of about 10 nM or lower. More preferably, the NGALmuteins can have an affinity measured by a KD of about 1 nM or lower.

In some embodiments, an hNGAL mutein of the disclosure includes at oneor more positions corresponding to position 28, 36, 40-41, 49, 52, 65,68, 70, 72-73, 75-77, 79, 81, 87, 96, 98, 100, 103, 106, 114, 120, 125,127, 134 and 175 of the linear polypeptide sequence of the mature hNGAL(SEQ ID NO: 43) a substitution.

In particular embodiments, a lipocalin mutein of the disclosurecomprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, or even more, substitution(s) at a sequenceposition corresponding to sequence position 28, 36, 40-41, 49, 52, 65,68, 70, 72-73, 75-77, 79, 81, 87, 96, 98, 100, 103, 106, 114, 120, 125,127, 134 and 175 of the linear polypeptide sequence of the mature hNGAL(SWISS-PROT Data Bank Accession Number P80188; SEQ ID NO: 43).Preferably, it is envisaged that the disclosure relates to a lipocalinmutein which comprises, in addition to one or more substitutions atpositions corresponding to positions 36, 87 and/or 96 of the linearpolypeptide sequence of the mature human NGAL, at one or more positionscorresponding to positions position 28, 36, 40-41, 49, 52, 65, 68, 70,72-73, 75-77, 79, 81, 87, 96, 98, 100, 103, 106, 114, 120, 125, 127, 134and 175 of the linear polypeptide sequence of the mature hNGAL asubstitution.

In some still further embodiments, the disclosure relates to apolypeptide, wherein said polypeptide is an hNGAL mutein, in comparisonwith the linear polypeptide sequence of the mature hNGAL, comprising atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, or even more, mutated amino acid residues at the sequencepositions position 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 75-77, 79,81, 87, 96, 98, 100, 103, 106, 114, 120, 125, 127, 134 and 175, andwherein said polypeptide binds IL-23p19, in particular human IL-23p19.

In some embodiments, an IL-23p19-binding hNGAL mutein of the disclosureincludes, at any one or more of the sequence positions 28, 36, 40-41,49, 52, 65, 68, 70, 72-73, 75-77, 79, 81, 87, 96, 98, 100, 103, 106,114, 120, 125, 127, 134 and 175 of the linear polypeptide sequence ofthe mature hNGAL (SEQ ID NO: 43), one or more of the following mutatedamino acid residues: Gln 28→His; Leu 36→Giu; Ala 40→Leu; Ile 41→Leu; Gln49→Arg; Tyr 52→Thr; Asn 65→Asp; Ser 68→Arg; Leu 70→Giu; Arg 72→Gly; Lys73→Ala or Via; Lys 75→Thr; Asp 77→Lys; Trp 79→Gln or Arg; Arg 81→Gly;Asn 96→Gly; Lys 98→Glu; Tyr 100→Met; Leu 103→Met; Tyr 106→Phe; Asn114→Asp; Met 120→Ile; Lys 125→Tyr; Ser 127→Tyr; and Lys 134→Giu. In someembodiments, an hNGAL mutein of the disclosure includes two or more,such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, even more or all mutated aminoacid residues at these sequence positions of the mature hNGAL.

Additionally, an IL-23p19-binding hNGAL mutein according to thedisclosure may also comprise the following substitution: Cys 87→Ser.Furthermore, an IL-23p19-binding hNGAL mutein according to thedisclosure may also comprise the following substitution: Cys 76→Tyr orArg. Furthermore, an IL-23p19-binding hNGAL mutein according to thedisclosure may also comprise the following substitution: Cys 175→Ala.

In some additional embodiments, an hNGAL mutein of the disclosure, whichbinds to IL-23p19 includes the one of following sets of amino acidreplacements in comparison with the linear polypeptide sequence of themature hNGAL:

-   -   (a) Gln 28→His; Leu 36→Giu; Ala 40-Leu; Ile 41→Leu; Gln 49→Arg;        Tyr 52→Thr; Asn 65→Asp; Ser 68→Arg; Leu 70→Giu; Arg 72→Gly; Lys        73→Ala; Lys 75→Thr; Cys 76→Tyr; Asp 77→Lys; Trp 79→Gln; Arg        81→Gly; Asn 96→Gly; Lys 98→Giu; Tyr 100→Met; Leu 103→Met; Tyr        106→Phe; Met 120→Ile; Lys 125→Tyr; Ser 127→Tyr; and Lys 134→Giu;    -   (b) Gln 28→His; Leu 36→Giu; Ala 40-Leu; Gln 49→Arg; Tyr 52→Thr;        Asn 65→Asp; Ser 68→Arg; Leu 70→Giu; Arg 72→Gly; Lys 73→Via; Lys        75→Thr; Cys 76→Arg; Asp 77→Lys; Trp 79→Arg; Arg 81→Gly; Asn        96→Gly; Tyr 100⊖Met; Leu 103→Met; Tyr 106→Phe; Lys 125→Tyr; Ser        127→Tyr; and Lys 134→Giu; or    -   (c) Gln 28→His; Leu 36→Giu; Ala 40-Leu; Ile 41→Leu; Gln 49→Arg;        Tyr 52→Thr; Asn 65→Asp; Ser 68→Arg; Leu 70→Giu; Arg 72→Gly; Lys        73→Val; Lys 75→Thr; Cys 76→Tyr; Asp 77→Lys; Trp 79→Gln; Arg        81→Gly; Asn 96→Gly; Tyr 100→Met; Leu 103→Met; Tyr 106→Phe; Asn        114→Asp; Lys 125→Tyr; Ser 127→Tyr; and Lys 134→Giu.

In the residual region, i.e. the region differing from sequencepositions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 75-77, 79, 81, 87,96, 98, 100, 103, 106, 114, 120, 125, 127, 134 and 175, an hNGAL muteinof the disclosure may include the wild type (natural) amino acidsequence outside the mutated amino acid sequence positions.

In further particular embodiments, a lipocalin mutein according to thecurrent disclosure comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 2 and 45-46 or a fragment or variantthereof.

The amino acid sequence of an IL-23p19-binding hNGAL mutein of thedisclosure may have a high sequence identity, such as at least 70%, atleast 75%, at least 80%, at least 82%, at least 85%, at least 87%, atleast 90% identity, including at least 95% identity, to a sequenceselected from the group consisting of SEQ ID NOs: 2 and 45-46.

The disclosure also includes structural homologues of an hNGAL muteinhaving an amino acid sequence selected from the group consisting of SEQID NOs: 2 and 45-46, which structural homologues have an amino acidsequence homology or sequence identity of more than about 60%,preferably more than 65%, more than 70%, more than 75%, more than 80%,more than 85%, more than 90%, more than 92% and most preferably morethan 95% in relation to said hNGAL mutein.

In some particular embodiments, the present disclosure provides alipocalin mutein that binds IL-23p19 with an affinity measured by aK_(D) of about 1 nM or lower, wherein the lipocalin mutein has at least90% or higher, such as 95%, identitiy to the amino acid sequence of SEQID NO: 2.

2. Applications of Lipocalin Muteins with Binding-Affinity forInterleukin-23p19 (IL-23p19)

Interleukin-23 (IL-23) is a heterodimeric cytokine composed of a uniquesubunit, p19 (herein referred to interchangeably as “IL-23p19”), and thep40 subunit, which is shared with interleukin-12 (IL-12) (Oppmann,Immunity 13:115 (2000)). IL-23 has been found to stimulate theproduction and/or maintenance of IL-17A and IL-17F from activated CD4 Tcells in what has now been termed as a “new” T-helper (Th) subset,designated Thl 7. A review of IL-23 cytokine and receptor biology isreviewed in Holscher, Curr. Opin. Invest. Drugs 6:489 (2005) andLangrish et al. Immunol Rev. 202:96 (2004). Similar to Thl and Th2lineages, Thl7 cells have most likely evolved to provide adaptiveimmunity to specific classes of pathogens, such as extracellularbacteria. However, inappropriate Th 17 responses have been stronglyimplicated in a growing list of autoimmune disorders, including multiplesclerosis, rheumatoid arthritis, inflammatory bowel disease, andpsoriasis.

In this regard, IL-23 promotes a distinct CD4 T cell activation statecharacterized by the production of interleukin-17 (J. Biol. Chem.278:1910-191 (2003); see also Langrish et al). IL-23 drives a pathogenicT cell population that induces autoimmune inflammation (J. Exp. Med.201: 233-240 (2005); Starnes et al. “Cutting edge: IL-17F, a novelcytokine selectively expressed in activated T cells and monocytes,regulates angiogenesis and endothelial cell cytokine production” J.Immunol. 167:4137-4140 (2001)).

Numerous possible applications for the muteins with binding-affinity forIL-23p19 of the disclosure, therefore, exist in medicine. In one furtheraspect, the disclosure relates to the use of such a mutein disclosed fordetecting IL-23p19 in a sample as well as a respective method ofdiagnosis.

The present disclosure also involves the use of one or more muteins withbinding-affinity for IL-23p19 as described for complex formation withIL-23p19.

Therefore, in another aspect of the disclosure, the disclosed muteinsare used for the detection of IL-23p19. Such use may include the stepsof contacting one or more said muteins, under suitable conditions, witha sample suspected of containing IL-23p19, thereby allowing formation ofa complex between the muteins and IL-23p19, and detecting the complex bya suitable signal.

The detectable signal can be caused by a label, as explained above, orby a change of physical properties due to the binding, i.e. the complexformation, itself. One example is plasmon surface resonance, the valueof which is changed during binding of binding partners from which one isimmobilized on a surface such as a gold foil.

The muteins disclosed herein may also be used for the separation ofIL-23p19. Such use may include the steps of contacting one or more saidmuteins, under suitable conditions, with a sample supposed to containIL-23p19, thereby allowing formation of a complex between the muteinsand IL-23p19, and separating the complex from the sample.

In the use of the disclosed muteins for the detection of IL-23p19 aswell as the separation of IL-23p19, the muteins and/or IL-23p19 or adomain or fragment thereof may be immobilized on a suitable solid phase.

Accordingly, the presence or absence of a molecule such as IL-23p19,e.g., in a sample, as well as its concentration or level may bedetermined.

In still another aspect, the present disclosure features a diagnostic oranalytical kit comprising a mutein with binding-affinity for IL-23p19according to the disclosure.

In addition to their use in diagnostics, in yet another aspect, thedisclosure encompasses the use of such a mutein of the disclosure or acomposition comprising such mutein for the binding of IL-23p19 in asubject and/or inhibiting the binding of IL-23 to its receptor in asubject.

In still another aspect, the present disclosure features a method ofbinding IL-23p19 in a subject, comprising administering to said subjectan effective amount of one or more lipocalin muteins withbinding-affinity for IL-23p19 of the disclosure or of one or morecompositions comprising such a mutein.

In still another aspect, the present disclosure involves a method forinhibiting the binding of IL-23 to its receptor in a subject, comprisingadministering to said subject an effective amount of one or morelipocalin muteins with binding-affinity for IL-23p19 of the disclosureor of one or more compositions comprising such a mutein.

C. Compositions Comprising an IL-17A Binding Lipocalin Mutein and/or anIL-23p19 Binding Lipocalin Mutein and Uses of the Lipocalin Muteins

IL-17A and IL-23 are cytokines involved in inflammation. Humaninterleukin-17A (also known as “IL-17”, including IL-17 A/A and IL-17A/F) is a cytokine which stimulates the expression of interleukin-6(IL-6), intracellular adhesion molecule 1 (ICAM-I), interleukin-8(IL-8), granulocyte macrophage colony-stimulating factor (GM-CSF), andprostaglandin E2 expression, and plays a role in the preferentialmaturation of CD34+ hematopoietic precursors into neutrophils (Yao etal, J. Immunol 755:5483 (1995); Fossiez et al, J. Exp. Med. 183:2593(1996)). Human interleukin-23 (also known as “IL-23”) is a cytokinewhich has been reported to promote the proliferation of T cells, inparticular memory T cells.

Both IL-17A (including IL-17A in complex with IL-17F, also termed asIL-17 A/F) and IL-23 have been reported to play important roles in manyautoimmune diseases, such as multiple sclerosis, rheumatoid arthritis,Crohn's disease, and psoriasis. Both IL-23 and IL-17A are overexpressedin the central nervous system of humans with multiple sclerosis and inmice undergoing an animal model of multiple sclerosis, experimentalautoimmune encephalomyelitis (EAE). The overexpression is observed inmice when the EAE is induced by either myelinoligodendrocyteglycoprotein (MOG) 35-55 peptide- or proteolipid peptide (PLP).Furthermore, neutralization of either IL-23p19 or IL-17A results inamelioration of EAE symptoms in mice (Park et al., Immunol 6:1133(2005); Chen et al., J Clin Invest. 116:1317 (2006)).

It has also been demonstrated that IL-17A and Th17 cells can be producedfrom IL-23-independent sources, and the in vivo development of an IL-17effector response has been shown to be IL-23-independent (Mangan et al.,Nature 441:231 (2006)). Neutralization of IL-23 would theoreticallyeliminate existing IL-17A producing cells, but would not completelyprevent the development of new Th17 cells.

The present disclosure, therefore, concerns the binding of both of theseproinflammatory cytokines, IL-17A and IL-23p19, since binding both IL-23(via p19) and IL-17A is more effective therapeutically thanneutralization of IL-23p19 alone or IL-17A alone and thus, beneficialfor the effective treatment of inflammatory diseases.

Although antibodies against IL-17A and/or IL-23p19 have been described,these antibody-based approaches still have a number of serious drawbackssuch as the necessity of complex mammalian cell production systems, adependency on disulfide bond stability, the tendency of some antibodyfragments to aggregate, limited solubility and last but not least, theymay elicit undesired immune responses even when humanized. There is anunmet need to, therefore, to develop small globular proteins such aslipocalins as scaffolds for the generation of a novel class of IL-17A orIL-23p19 binding proteins, e.g. lipocalin muteins with binding-affinityfor IL-17A or IL-23p19.

Accordingly, it is an object of the present disclosure to provide humanlipocalin muteins that bind IL-17A (including IL-17 A/A and IL-17 A/F)and/or IL-23p19 and can be used in pharmaceutical applications. Thedisclosure also provides one or more compositions comprising suchlipocalin muteins and, optionally, one or more pharmaceutically ordiagnostically acceptable excipients (e.g. adjuvants, diluents orcarriers). Lipocalin muteins of the disclosure as well as compositionsthereof may be used in methods of detecting IL-17A (including IL-17 A/Aand IL-17 A/F) and/or IL-23p19 in a sample or in methods of binding ofIL-17A (including IL-17 A/A and IL-17 A/F) and/or IL-23p19 in a subject.

As discussed above, binding IL-17A (including IL-17 A/A and IL-17 A/F)and IL-23p19 concomitantly with lipocalin muteins specific for IL-17A(including IL-17 A/A and IL-17 A/F) or IL-23p19, respectively, couldovercome some of the hypoxia-mediated effects that binding IL-17A(including IL-17 A/A and IL-17 A/F) alone or binding IL-23p19 alone,respectively, might induce. The present disclosure, therefore,encompasses use of (i) a first lipocalin mutein specific for IL-17A and(ii) a second lipocalin mutein specific for IL-23p19, for the binding ofIL-17A and IL-23p19 in a subject. Such use includes a step ofadministering to a subject an effective amount of (i) a first lipocalinmutein specific for IL-17A and (ii) a second lipocalin mutein specificfor IL-23p19.

In the context of the present disclosure, the lipocalin mutein specificfor IL-17A can binds to IL-17A that exists as a homodimer (i.e. IL-17A/A), but it can also binds to IL-17A that exists as a heterodimercomplexed with the homolog IL-17F to form heterodimeric IL-17 A/F. Inone preferred embodiment, said lipocalin mutein binds to IL-17A andIL-17F complex.

The first lipocalin mutein and the second lipocalin mutein may beadministered in combination, including concurrently, concomitantly or inseries. In some embodiments, the first lipocalin mutein and the secondlipocalin mutein may be included in a composition that may beadministered. The composition may include an effective amount of thefirst and the second lipocalin mutein as active ingredients, inassociation with at least one pharmaceutically acceptable adjuvant,diluent or carrier. The first lipocalin mutein and the second lipocalinmutein may also be administered independent from each other, includingat individual intervals at independent points of time.

In some embodiments, the present disclosure also relates to acomposition comprising a first lipocalin mutein specific for IL-17A and(ii) a second lipocalin mutein specific for IL-23p19, which compositioncan be used in a method of binding of IL-17A and IL-23p19 e.g. in asubject. In addition, such composition may be used in a method ofdetecting IL-17A (including IL-17 A/A and IL-17 A/F) and IL-23p19 e.g.in a sample.

In some other embodiments, the present disclosure relates to acombination of a first lipocalin mutein and a second lipocalin mutein.One of these lipocalin muteins can bind to IL-17A as a given non-naturaltarget with detectable affinity. The other lipocalin mutein can bind toIL-23p19 as a given non-natural target with detectable affinity. Therespective lipocalin mutein thus binds to IL-17A or to IL-23p19,respectively, as a given non-natural target. The term “non-naturaltarget” refers to a compound, which does not bind to the correspondinglipocalin under physiological conditions. For example, the firstlipocalin mutein can bind to IL-17A and the second lipocalin mutein canbind to IL-23p19, or vice versa. The combination of the first lipocalinmutein and the second lipocalin mutein may be provided in various forms.

In some embodiments, the lipocalin mutein specific for IL-17A as used inthe disclosure is able to bind IL-17A with detectable affinity, i.e.with a dissociation constant of at least 200 nM, including about 100 nM,about 50 nM, about 25 nM or about 15 nM. In some embodiments, thelipocalin mutein specific for IL-23p19 as used in the disclosure is ableto bind IL-23p19 with detectable affinity, i.e. with a dissociationconstant of at least 200 nM including about 100 nM, about 50 nM, about25 nM or about 15 nM. In some further preferred embodiments, a lipocalinmutein of the combination according to the disclosure binds IL-17A orIL-23p19, respectively, with dissociation constant for IL-17A orIL-23p19 of at least about 10 nM, about 1 nM, about 0.1 nM, about 10 pM,or even lower. The present disclosure, thus, provides a combination of(i) a mutein of a lipocalin that has a particularly high affinity toIL-17A and (ii) a mutein of a lipocalin that has a particularly highaffinity to IL-23p19.

In some embodiments, the lipocalin muteins with a detectable affinityfor IL-17A are muteins of human tear lipocalin. These and furtherdetails on lipocalin muteins with a detectable affinity for IL-17A canbe found in Section A of the current disclosure.

In a particularly preferred embodiment, a lipocalin mutein that isspecific for IL-17A is shown in SEQ ID NO: 1.

In some embodiments, the lipocalin muteins with a detectable affinityfor IL-23p19 are muteins of human tear lipocalin or muteins of humanneutrophil gelatinase associated lipocalin. These and further details oflipocalin muteins with a detectable affinity for IL-23p19 have beendisclosed in in Section B of the current disclosure.

In a particular preferred embodiment, the lipocalin mutein that isspecific for IL-23p19 is shown in any one of SEQ ID NOs: 2, 45 and 46.

In still another aspect, the present disclosure features a method ofbinding IL-17A and IL-23 in a subject comprising administering to saidsubject an effective amount of (i) a first lipocalin mutein specific forIL-17A and (ii) a second lipocalin mutein specific for IL-23p19.

In still another aspect, the present disclosure involves a method forinhibiting the binding of IL-17A and IL-23 to their respectivereceptor(s) in a subject comprising administering to said subject aneffective amount of (i) a first lipocalin mutein specific for IL-17A and(ii) a second lipocalin mutein specific for IL-23p19.

The present disclosure also involves the use of (i) a first lipocalinmutein specific for IL-17A and (ii) a second lipocalin mutein specificfor IL-23p19 for complex formation with IL-17A and IL-23p19.

Therefore, in another aspect of the disclosure, the disclosed muteinscan be used for the detection of IL-17A and IL-23p19. Such use mayinclude the steps of contacting two or more said muteins, under suitableconditions, with a sample suspected of containing IL-17A and IL-23p19,thereby allowing formation of a complex between the muteins and IL-17Aor between the muteins and IL-23p19, respectively, and detecting thecomplex by a suitable signal.

The detectable signal can be caused by a label, as explained above, orby a change of physical properties due to the binding, i.e. the complexformation, itself. One example is plasmon surface resonance, the valueof which is changed during binding of binding partners from which one isimmobilized on a surface such as a gold foil.

The muteins disclosed herein may also be used for the separation ofIL-17A and IL-23p19. Such use may include the steps of contacting one ormore said muteins, under suitable conditions, with a sample supposed tocontain IL-17A and IL-23p19, thereby allowing formation of a complexbetween the muteins and IL-17A or between the muteins and IL-23,respectively, and separating the complex from the sample.

In the use of the disclosed muteins for the detection of IL-17A andIL-23p19 as well as the separation of IL-17A and IL-23p19, the muteinsand/or IL-17A and IL-23p19 or a domain or fragment thereof may beimmobilized on a suitable solid phase.

Accordingly, the presence or absence of IL-17A and/or IL-23p19, e.g., ina sample, as well as their concentration or level may be determined.

In another aspect, the disclosure provides for a kit of parts. The kitincludes a first and a second container. The first container includesthe first lipocalin mutein and the second container includes the secondlipocalin mutein. In one aspect, the disclosure relates to a kit thatincludes, in one or more containers, separately or in admixture, alipocalin mutein specific for IL-17A. In yet another aspect, thedisclosure also relates to a kit that includes, in one or morecontainers, separately or in admixture, a lipocalin mutein specific forIL-23p19. In some embodiments, the disclosure relates to a kit thatincludes, in one or more containers, separately or in admixture, alipocalin mutein specific for IL-17A and a lipocalin mutein specific forIL-23p19. In some further preferred embodiments, the kid comprises afirst container that includes a first lipocalin mutein specific forIL-17A and a second container that includes a second lipocalin muteinspecific for IL-23p19. In some embodiments the kit further includesintegrally thereto or as one or more separate documents, informationpertaining to the contents or the kit and the use of the lipocalinmuteins. The kit may include in some embodiments one or morecompositions that are formulated for reconstitution in a diluent. Such adiluent, e.g. a sterile diluent, may also be included in the kit, forexample within a container.

D. Fusion Proteins with Binding Affinity for IL-17A and/or IL-23p19 andUses Thereof

In one aspect, the present disclosure relates to a fusion proteincomprising at least two subunits in any order: one subunit has bindingspecificity for IL-17A and another subunit has binding specificity forIL-23p19.

For example, the present disclosure provides a fusion protein that hasprotein moieties with binding specificity for IL-17A (including IL-17A/A and IL-17 A/F) and IL-23p19, respectively. In this regard, onesubunit of said fusion protein may comprise a lipocalin mutein of thedisclosure specific for IL-17A (including IL-17 A/A and IL-17 A/F) whileanother subunit of said fusion protein may comprise a lipocalin muteinof the disclosure specific for IL-23p19.

In another aspect, the present disclosure is pertinent to a fusionprotein comprising at least two subunits, where each has a bindingspecificity for IL-17A (including IL-17 A/A and IL-17 A/F). In someembodiments, at least one subunit comprises a lipocalin mutein specificfor IL-17A. In some embodiments, the fusion protein has a bindingaffinity for IL-17A by a KD of about 1 nM or lower in an assayessentially described in Example 2. In some additional embodiments, thefusion protein is capable of inhibiting the binding of IL-17A to itsreceptor in a competition ELISA format essentially described in Example3 or in an assay essentially described in Example 5.

In some further embodiments, each of the two subunits comprises alipocalin mutein specific for IL-17 A/A. In some further embodiments,each of the two subunits comprises a lipocalin mutein specific for IL-17A/F. The two lipocalin muteins may have a different amino acid sequence.Hence, in some embodiment, the two lipocalin muteins bind to a differentepitope on IL-17A. In some other embodiments, however, the two lipocalinmuteins may be identical to each other. For example, such a fusionprotein may comprise two I amino acid sequences of SEQ ID NO: 1. In thisregard, the fusion protein may have the amino acid sequence shown in SEQID NO: 10, SEQ NO: 12 or SEQ ID NO: 13.

In some embodiments, a fusion protein of the disclosure having twosubunits that have binding specificity to IL-17A (including IL-17 A/Aand IL-17 A/F) may exhibit a higher potency than a single subunit, dueto an avidity effect of the two subunits, which is brought about by thedimeric nature of the target (e.g. IL17 A/A). In this regard, the fusionprotein can be a bivalent fusion protein. In still another aspect, thepresent disclosure also encompasses a fusion protein comprising at leasttwo subunits that have binding specificity for IL-23p19. In someembodiments, at least one subunit comprises a lipocalin mutein specificfor IL-23p19. In some embodiments, the fusion protein has a bindingaffinity for IL-23p19 by a KD of about 10 nM or lower in an assayessentially described in Example 7. In some additional embodiments, thefusion protein is capable of inhibiting the binding of IL-23 to itsreceptor in a competition ELISA format essentially described in Example8 or Example 14 or in an assay essentially described in Example 10 orExample 15.

In some further embodiments, each of the two subunits comprises alipocalin mutein specific for IL-23p19. The two lipocalin muteins mayhave a different amino acid sequence. Hence, in some embodiment, the twolipocalin muteinsbind to a different epitope on IL-23p19. In some otherembodiments, however, the two lipocalin muteins may be identical to eachother.

In one further aspect, the present application discloses a fusionprotein comprising (i) the Fc part of an immunoglobulin, including afull-length human antibody, such as an IgG antibody, and (ii) alipocalin mutein specific for IL-17A.

In another aspect, the present application discloses a fusion proteincomprising (i) the Fc part of an immunoglobulin, including a full-lengthhuman antibody, such as an IgG antibody, and (ii) a lipocalin muteinspecific for IL-23p19.

Exemplary lipocalin muteins specific for IL-17A (including IL-17 A/A andIL-17 A/F) include those disclosed in Section A of the currentdisclosure. In a particularly preferred embodiment, the lipocalin muteinis shown in SEQ ID NO: 1.

Exemplary lipocalin muteins specific for IL-23p19 include thosedisclosed in Section B of the current disclosure. In a particularlypreferred embodiment, the lipocalin mutein is shown in any one of theSEQ ID NOs: 2, 45 and 46.

In some particular embodiments, the lipocalin mutein can be linked, forexample, via a peptide bond, to the C-terminus and/or the N-terminus ofthe Fc part of a human antibody (see FIG. 11). In a particularembodiment, a fusion protein of the disclosure may comprise a lipocalinmutein attached to the Fc part of an IgG antibody. In this regard, oneof such fusion proteins comprises the amino acid sequences shown in SEQID NO: 16.

In a still preferred embodiment, a fusion protein of the disclosurecomprises the amino acids shown in SEQ ID NO: 11, SEQ ID NO: 12 or SEQID NO: 13.

In a related embodiment, one or more fusion proteins of the disclosureare capable of inhibiting the binding of IL-17A and the binding of IL-23to their respective receptor(s). In some further embodiments, one ormore fusion proteins of the disclosure are capable of engaging IL-17Aand IL-23p19 simultaneously, and, hence, thereby are capable ofinhibiting the binding of IL-17A and the binding of IL-23 to theirrespective receptor(s) at the same time.

In this aspect, the present disclosure relates to a fusion proteincomprising at least two subunits in any order, including one subunitcomprises a lipocalin mutein specific for IL-17A (including IL-17 A/Aand IL-17 A/F) and one subunit comprises a lipocalin mutein specific forIL-23p19. In some further embodiments, the fusion protein may contain anadditional subunit, which subunit comprises a lipocalim mutein specificfor IL-17A (including IL-17 A/A and IL-17 A/F) or IL-23p19. In someembodiments, two IL-17A-specific lipocalin muteins, as included in twodifferent subunits of the fusion protein, may bind to different epitopeson the IL-17A target; alternatively, the two IL-17A-specific lipocalinmuteins, as included in two different subunits of the fusion protein,may have the same amino acid sequence and, hence, have the specificityfor the same epitope on the IL-17A target. A fusion protein of thedisclosure having two subunits binding to IL-17A may exhibit a strongerbinding to IL-17A than a fusion protein having only one subunit bindingto IL-17A, due to an avidity effect brought about by the dimeric natureof the target. Likewise, two IL-23p19-specific lipocalin muteins, asincluded in two different subunits of the fusion protein, may bind todifferent epitopes on the IL-23p19 target; alternatively, the twoIL-23p19-specific lipocalin mutein, as included in two differentsubunits of the fusion protein, may have the same amino acid sequenceand, hence, the specificity for the same epitope on the IL-23p19 target.A fusion protein may also include a linker that links one subunit toanother subunit.

In some embodiments, one subunit of a fusion protein of the disclosurecomprises a lipocalin mutein disclosed in Section A of the currentdisclosure. In a particularly preferred embodiment, the subunitcomprises a lipocalin mutein shown in SEQ ID NO: 1.

In some embodiments, one subunit of a fusion protein of the disclosurecomprises a lipocalin mutein disclosed in in Section B of the currentdisclosure. In a particular preferred embodiment, the subunit comprisesa lipocalin mutein shown in any one of the SEQ ID NOs: 2, 45 and 46.

In some embodiments, a fusion protein of the disclosure comprises alipocalin mutein disclosed in Section A as well as a lipocalin muteindisclosed in in Section B.

In a particular embodiment, a fusion protein of the disclosure comprisesthe amino acid sequence shown in SEQ ID NO: 3.

In a particular embodiment, a fusion protein of the disclosure comprisesthe amino acid sequence shown in SEQ ID NO: 4.

In a still preferred embodiment, a fusion protein of the disclosurecomprises the amino acids shown in SEQ ID NO: 12 or SEQ ID NO: 13.

In another aspect, the present application discloses a fusion proteincomprising at least two subunits, wherein one subunit has bindingspecificity for IL-17A or IL-23p19 and another subunit contains analbumin binding domain (ABD) or an albumin binding peptide. In someembodiments, the subunit has binding specificity for IL-17A or IL-23p19comprises a lipocalin mutein specific for IL-17A or IL-23p19 of thedisclosure. Furthermore, the fusion protein may comprise in any order(i) one subunit specific for IL-17A, (ii) one subunit specific forIL-23p19 and (iii) one subunit that contains a bacterial albumin bindingdomain. In some embodiments, the subunit has binding specificity forIL-17A comprises a lipocalin mutein specific for IL-17A of thedisclosure. In some other embodiments, the subunit has bindingspecificity for IL-23p19 comprises a lipocalin mutein specific forIL-23p19 of the disclosure.

In some embodiments, the albumin binding domain (ABD) may be astreptococcal protein G (König, T., & Skerra, A. (1998) J. Immunol.Methods 218, 73-83) or a fragment thereof, e.g. as shown in SEQ ID NO:14. In some other embodiments, the albumin binding peptide is a humanserum albumin binding peptide derived from the albumin binding domain ofstreptococcal protein G, for example, as disclosed in PCT applicationWO2012/004384, which is incorporated by reference its entirety herein.In some still preferred embodiments, the albumin binding peptidecomprises the amino acid sequence shown in SEQ ID NO: 15.

In particular, the present disclosure provides a fusion protein, whichis capable of binding to both human serum albumin (HSA) and IL-23p19simultaneously, for example, comprising the amino acid sequence shown inSEQ ID NO: 7.

The present disclosure provides a fusion protein, which is capable ofbinding to both HSA and IL-17A simultaneously, for example, comprisingthe amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 10. In somestill preferred embodiment, such fusion protein may include twoIL-17A-specific lipocalin muteins, for example, comprising the aminoacid sequence shown in SEQ ID NO: 10.

In addition, the present application features a fusion protein, which iscapable of binding to all HSA, IL-17A and IL-23p19 simultaneously, forexample, when the fusion protein is measured in an assay essentiallydescribed in Example 12, In some further embodiments, the fusion proteincomprises the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 6 orof SEQ ID NO: 9. In some still preferred embodiment, such fusion proteinmay include two IL-17A-specific lipocalin muteins, for example,comprising the amino acid sequence shown in SEQ ID NO: 6.

In an additional aspect, the present disclosure relates to a fusionprotein comprising at least two subunits in any order: one subunit hasbinding specificity for IL-17A or has binding specificity for IL-23p19and another subunit has binding specificity for TNF, for example,comprising a TNF-inhibiting protein. Tumor necrosis factors (or the TNFfamily) refer to a group of cytokines that can cause cell death(apoptosis) such as TNF-alpha (TNF-α) and Lymphotoxin-alpha. ExemplaryTNF inhibitors include adalimumab, infliximab, etanercept, certolizumabpegol and golimumab. In some further embodiments, the TNF-specificsubunit comprises an anti-TNF-α antibody, such as the antibody of SEQ IDNOs: 61 and 62. In some additional embodiments, the subunit has bindingspecificity for IL-17A or has binding specificity for IL-23p19 comprisesa lipocalin mutein of the disclosure such as the lipocalin of SEQ ID NO:1 or the lipocalin of SEQ ID NO: 2. In some still further embodiments,the fusion proteins comprises the amino acid sequences of SEQ ID NOs: 63and 62 or the amino acid sequences of SEQ ID NOs: 64 and 62.

In some embodiments, the fusion protein is capable of binding 17A and,in some preferred embodiments, may have an average EC50 value at leastas good as or superior to the average EC50 value of the lipocalin muteinas included in the fusion protein, for example, when the fusion proteinand the lipocalin mutein are measured in an assay essentially describedin Example 16. In some embodiments, the fusion protein is capable ofbinding IL-23 and in some preferred embodiments, may have an averageEC50 value at least as good as or superior to the average EC50 value ofthe lipocalin mutein as included in the fusion protein, for example,when the fusion protein and the lipocalin mutein are measured in anassay essentially described in Example 17. In some embodiments, thefusion protein is capable of binding TNF-α and, in some preferredembodiments, may have an average EC50 value at least as good as orsuperior to the average EC50 value of the antibody as included in thefusion protein, when the antibody and the fusion protein are measured inan assay essentially described in Example 18. In some furtherembodiments, the fusion protein may be capable of binding to all TNF-α,IL-17A and IL-23p19 simultaneously, for example, when the fusion proteinis measured in an assay essentially described in Example 19.

In some embodiments, a fusion protein of the disclosure has a bindingaffinity for IL-17A (including IL-17 A/A and IL-17 A/F) measured by a KDof about 1 nM or lower. More preferably, said fusion protein may have anaffinity measured by a KD of 0.1 nM or lower. In some furtherembodiments, the IL-17A-binding moiety (or moieties) of a fusion proteinof the disclosure may have a binding affinity or inhibiting ability forIL-17A (including IL-17 A/A and IL-17 A/F) as good as that of suchmoiety as a stand-alone polypeptide (see Table 1).

In some embodiments, a fusion protein of the disclosure has a bindingaffinity for IL-23p19 measured by a KD of about 10 nM or lower. Morepreferably, said fusion protein may have an affinity measured by a KD ofabout 1 nM or lower. In some further embodiments, the IL-23p19-bindingmoiety (or moieties) of a fusion protein of the disclosure may have abinding affinity or inhibiting ability for IL-23p19 as good as that ofsuch moiety as a stand-alone polypeptide (see Table 1 below).

Table 1: provides an overview of the activity of individual lipocalinmuteins SEQ ID NO: 1 and SEQ ID NO: 2, compared to their fusion proteinsSEQ ID NO's: 3-13 in competition ELISA, surface plasmon resonance (SPR)and functional cell-based assays. Values were determined for interactionwith IL-17 and/or IL-23, depending on whether the respective constructcontains the IL-17A-binding lipocalin mutein SEQ ID NO: 1, theIL-23-binding lipocalin mutein SEQ ID NO: 2, or both. To determine theactivity towards IL-17 and IL-23, respectively, competition ELISAexperiments were carried out as described in Example 3 and/or Example 8,SPR experiment in reverse format—i.e. with the protein constructsimmobilised on the sensor chip—were carried out as described in Example2 and/or Example 6, and cell assays were based on either IL-17A-inducedG-CSF secretion (Example 5) and/or IL-23 induced Ba/F3 cellproliferation (Example 10). Note that the reverse format SPR experimentperformed to determine IL-23 affinity was carried out in the presence ofunphysiologically high concentrations of NaCl, and that the valuestherefore do not reflect the affinity to IL-23 under physiologicalconditions, but serve to determine whether the relative affinity toIL-23 is different in the SEQ ID NO: 2-containing fusion proteins of SEQID NO's: 3-13, compared to the individual mutein SEQ ID NO: 2. Table 1demonstrates that the IL-17A-binding activity of all fusions containingSEQ ID NO: 1 is at least as good as that of SEQ ID NO: 1 itself in allassay formats. SEQ ID NO: 1 can therefore flexibly be employed in anyfusion protein without activity loss. The IL-23-binding activity of allfusions containing SEQ ID NO: 2 is very close to that of SEQ ID NO: 2itself in all assay formats. SEQ ID NO: 2 can therefore flexibly beemployed in any fusion protein without significant activity loss.

Competition Biacore. G-CSF Competition Biacore. Cell ELISA rev. formatsecretion ELISA rev. format proliferation (IC50, IL17A) (Kd, IL17AF)(EC50, IL17A) (IC50, IL23) (Kd, IL23) (EC50, IL23) SEQ ID cf. Example 3cf. Example 2 cf. Example 5 cf. Example 8 cf. Example 7 cf. Example 10SEQ ID NO: 1 0.08 nM (0.06-0.09) 0.10 nM 0.13 nM (0.17/0.1) SEQ ID NO: 20.54 nM (0.26-0.83) 2.9 1.2 nM (1.7/0.7) SEQ ID NO: 8 0.10 nM(0.07-0.13) 0.10 nM SEQ ID NO: 7 1.11 nM (0.69-1.53) 2.4 SEQ ID NO: 100.05 nM (0.03-0.07) 0.04 nM 0.1 nM (0.15/0.04) SEQ ID NO: 3 0.04 nM(0.03-0.05) 0.10 nM 0.15 nM (0.21/0.09) 0.64 nM (0.49-0.79) 3.0 0.9 nM(1.1/0.6) SEQ ID NO: 4 0.07 nM (0.05-0.09) 0.08 nM 0.24 nM (0.20/0.29)0.99 nM (0.69-1.29) 2.0 2.8 nM (2.8/2.7) SEQ ID NO: 5 0.05 nM(0.03-0.06) 0.08 nM 0.15 nM (0.20/0.10) 1.09 nM (0.63-1.56) 1.7 n.d. SEQID NO: 9 0.05 nM (0.04-0.07) 0.09 nM 0.16 nM (0.19/0.12) 2.07 nM(0.91-3.22) 1.9 2.1 nM (2.4/1.7) SEQ ID NO: 6 0.04 nM (0.02-0.05) 0.10nM 0.05 nM (0.06/0.04) 0.97 nM (0.61-1.33) 1.9 0.8 nM (0.9/0.7) SEQ IDNO: 11 0.09 nM (0.06-0.12) 0.07 nM 0.06 ± 0.04 nM (n = 3) SEQ ID NO: 120.03 nM (0.01-0.04) 0.04 nM 0.05 ± 0.04 nM (n = 3) SEQ ID NO: 13 0.04 nM(0.03-0.05) n.d. 0.05 ± 0.04 nM (n = 3)

In a related embodiment, one or more fusion proteins of the disclosureare capable of inhibiting the binding of IL-17A to its receptor.

In a related embodiment, a fusion protein of the disclosure is capableof inhibiting the binding of IL-23 to its receptor.

In some embodiments, a fusion protein of the disclosure may also includea linker (e.g. a peptide bond) that covalently links a lipocalin muteinof the disclosure and another lipocalin mutein of the disclosure to eachother. This can be achieved, for example, by expression of the linkedlipocalin muteins as a single polypeptide connected by a peptide linker.A suitable peptide linker can be comprised of a stretch of amino acidsof arbitrary length containing any amino acids, e.g. as describedherein. A preferred linker design utilizes a repeated stretch of aminoacids of glycines and serines following the formula (GxSy)n, where x isthe number of glycine repeats and y the number of serine repeats in abuilding block that is repeated n times. The values of each of thevariables x, y, and n can range from 0 to 100, preferably from 0 to 10.Non-limiting examples are hereby provided with SEQ ID NOs: 18-20.

In some other embodiments, chemical methods of covalently linking may beapplied to link a lipocalin mutein of the disclosure to anotherlipocalin mutein of the disclosure. One example is the use ofbifunctional linkers that allow reactive chemistry between the linkerand an amino acid side chain, for example, between a maleimide and afree cysteine in a lipocalin mutein, or an activated carboxylic acidester and a primary amine in the lipocalin mutein. This includesreaction with non-natural amino acid side chains that may be includedduring protein expression, and which provide a functionality that can beselectively derivatised. In some still further embodiments, “click”chemistry, such as the cycloaddtion of an azide and an alkine, may beused to link one or more subunits of a fusion protein of the disclosure.

In some further preferred embodiments, a fusion protein of thedisclosure further comprises the amino acid sequence shown in any one ofSEQ ID NOs: 18-20.

In some further embodiments, one subunit comprising a lipocalin muteinof the disclosure may be, directly or via a chemical linker attached, toanother subunit comprising a lipocalin mutein of the disclosure in afusion protein as disclosed herein.

In some still further embodiments, a lipocalin mutein of the disclosurecan be fused either to the N- or C-terminus or to both the N- and theC-termini of another lipocalin mutein.

In some embodiments, each of the subunits as comprised in a fusionprotein of disclosure, stay thermostable (e.g. can resist a meltingtemperature at a T_(m) of at least 40° C.). In some embodiments, each ofsaid three subunits, comprised in a fusion protein of disclosure, arewith high cooperativity of unfolding with respect to one or more othersubunits (e.g. eliminate partial unfolding, and thus significantlyreducing their rate of degradation). This elimination of partialunfolding is termed “cooperative,” because unfolding is an all-or-noneprocess. In some further embodiments, one or more lipocalin muteins asincluded in the fusion protein can resist a melting temperature at a Tmof at least 50° C., at least 55° C., at least 60° C. or even higher. Insome still further embodiments, one or more HSA component as included inthe fusion protein can resist a melting temperature at a T_(m) of atleast 30° C., at least 35° C., at least 40° C. or even higher.

In some embodiments, the one or more fusion proteins of the disclosurecomprise multimers: e.g., tetramers, trimers or dimers of the lipocalinmuteins of the disclosure, wherein at least one lipocalin mutein isfused to at least one side (e.g. to the N-terminus) of another lipocalinmutein. In some further embodiments, multimeric fusion proteins may bepreferred to the corresponding monomeric fusion protein. For example, adimeric fusion protein of the disclosure binding to IL-17A may exhibit astronger binding to IL-17A due to an avidity effect brought about by thedimeric nature of the target.

In some further embodiment, one or more fusion proteins of thedisclosure result in the formation of “Duocalins” as described inSchlehuber, S., and Skerra, A. (2001), Duocalins, engineeredligand-binding proteins with dual specificity derived from the lipocalinfold. Biol. Chem. 382, 1335-1342, the disclosure of which is herebyincorporated by reference in its entirety.

In still another aspect, the disclosure encompasses the use of one ormore fusion proteins of the disclosure or of one or more compositionscomprising such proteins for the binding of IL-17A and/or IL-23p19 in asubject and/or inhibiting the binding of IL-17 and/or IL-23 to theirrespective receptor(s) in a subject.

In still another aspect, the present disclosure features a method ofbinding IL-17A and/or IL-23p19 in a subject, comprising administering tosaid subject an effective amount of one or more fusion proteins of thedisclosure or of one or more compositions comprising such proteins.

In still another aspect, the present disclosure involves a method forinhibiting the binding of IL-17 and/or IL-23 to their respectivereceptor(s) in a subject, comprising administering to said subject aneffective amount of one or more fusion proteins of the disclosure or ofone or more compositions comprising such proteins.

Fusion proteins of the disclosure may also include a signal sequence.Signal sequences at the N-terminus of a polypeptide direct thispolypeptide to a specific cellular compartment, for example theperiplasm of E. coli or the endoplasmatic reticulum of eukaryotic cells.A large number of signal sequences are known in the art. An illustrativesignal sequence for secretion a polypeptide into the periplasm of E.coli is the OmpA-signal sequence.

The present disclosure also involves the use of one or more fusionproteins of the disclosure for complex formation with IL-17A and/orIL-23p19.

Therefore, in another aspect of the disclosure, one or more fusionproteins of the disclosure can be used for the detection of IL-17Aand/or IL-23p19. Such use may include the steps of contacting one ormore fusion proteins of the disclosure, under suitable conditions, witha sample suspected of containing IL-17A and/or IL-23p19, therebyallowing formation of a complex between the proteins and IL-17A and/orbetween the proteins and IL-23p19, respectively, and detecting thecomplex by a suitable signal.

The detectable signal can be caused by a label, as explained above, orby a change of physical properties due to the binding, i.e. the complexformation, itself. One example is plasmon surface resonance, the valueof which is changed during binding of binding partners from which one isimmobilized on a surface such as a gold foil.

The one or more fusion proteins disclosed herein may also be used forthe separation of IL-17A and/or IL-23p19 from a sample that containsother substances. Such use may include the steps of contacting one ormore said fusion proteins, under suitable conditions, with a samplesupposed to contain IL-17A and/or IL-23p19, thereby allowing formationof a complex between the proteins and IL-17A and/or between the proteinsand IL-23, respectively, and separating the complex from the sample.

In the use of a disclosed fusion proteins for the detection of IL-17Aand/or IL-23p19 as well as the separation of IL-17A and/or IL-23p19, thefusion protein, IL-17A, IL-23p19 and/or a domain or fragment thereof maybe immobilized on a suitable solid phase.

Accordingly, the presence or absence of molecules such as IL-17A and/orIL-23p19, e.g., in a sample, as well as its concentration or level maybe determined.

In another aspect, the disclosure provides for a kit comprising at leastone fusion protein of the disclosure and one or more instructions forusing the kit.

In some embodiments the kit further includes integrally thereto or asone or more separate documents, information pertaining to the contentsor the kit and the use of the fusion proteins. The kit may include insome embodiments one or more fusion proteins of the disclosure that areformulated for reconstitution in a diluent. Such a diluent, e.g. asterile diluent, may also be included in the kit, for example within acontainer.

In some embodiments, the one or more fusion proteins of the disclosuremay be used in the treatment of several conditions where the suppressionof the immune response is desired such as Rheumatoid arthritis,Psoriatic arthritis, Ankylosing spondylitis, Crohn's disease, Ulcerativecolitis, Plaque psoriasis, and Juvenile idiopathic arthritis.

E. Lipocalin Mueteins and Fusion Proteins of the Disclosure

Lipocalins are proteinaceous binding molecules that have naturallyevolved to bind ligands. Lipocalins occur in many organisms, includingvertebrates, insects, plants and bacteria. The members of the lipocalinprotein family (Pervaiz, S., & Brew, K. (1987) FASEB J. 1, 209-214) aretypically small, secreted proteins and have a single polypeptide chain.They are characterized by a range of different molecular-recognitionproperties: their ability to bind various, principally hydrophobicmolecules (such as retinoids, fatty acids, cholesterols, prostaglandins,biliverdins, pheromones, tastants, and odorants), their binding tospecific cell-surface receptors and their formation of macromolecularcomplexes. Although they have, in the past, been classified primarily astransport proteins, it is now clear that the lipocalins fulfill avariety of physiological functions. These include roles in retinoltransport, olfaction, pheromone signalling, and the synthesis ofprostaglandins. The lipocalins have also been implicated in theregulation of the immune response and the mediation of cell homoeostasis(reviewed, for example, in Flower, D. R. (1996) Biochem. J. 318, 1-14and Flower, D. R. et al. (2000) Biochim. Biophys. Acta 1482, 9-24).

The lipocalins share unusually low levels of overall sequenceconservation, often with sequence identities of less than 20%. In strongcontrast, their overall folding pattern is highly conserved. The centralpart of the lipocalin structure consists of a single eight-strandedanti-parallel β-sheet closed back on itself to form a continuouslyhydrogen-bonded β-barrel. This β-barrel forms a central cavity. One endof the barrel is sterically blocked by the N-terminal peptide segmentthat runs across its bottom as well as three peptide loops connectingthe β-strands. The other end of the β-barrel is open to the solvent andencompasses a target-binding site, which is formed by four flexiblepeptide loops. It is this diversity of the loops in the otherwise rigidlipocalin scaffold that gives rise to a variety of different bindingmodes each capable of accommodating targets of different size, shape,and chemical character (reviewed, e.g., in Flower, D. R. (1996), supra;Flower, D. R. et al. (2000), supra, or Skerra, A. (2000) Biochim.Biophys. Acta 1482, 337-350).

A lipocalin mutein according to the present disclosure may be a muteinof any chosen lipocalin. Examples of suitable lipocalins (also sometimesdesignated as “protein ‘reference’ scaffolds” or simply “scaffolds”) ofwhich a mutein may be used include, but are not limited to, tearlipocalin (lipocalin-1, von Ebner gland protein), retinol bindingprotein, neutrophil, lipocalin-type prostaglandin D-synthase,β-lactoglobulin, bilin-binding protein (BBP), apolipoprotein D (APO D),neutrophil gelatinase associated lipocalin (NGAL), tear lipocalin (Tlc),α2-microglobulin-related protein (A2m), 24p3/uterocalin (24p3), vonEbners gland protein 1 (VEGP 1), von Ebners gland protein 2 (VEGP 2),and Major allergen Can f1 precursor (ALL-1). In related embodiments, thelipocalin mutein is selected from the group consisting of humanneutrophil gelatinase associated lipocalin (NGAL), human tear lipocalin(Tlc), human apolipoprotein D (APO D) and the bilin-binding protein ofPieris brassicae.

When used herein in the context of the lipocalin muteins of the presentdisclosure that bind to IL-17A or IL-23p19, the term “specific for”includes that the lipocalin mutein is directed against, binds to, orreacts with IL-17A or IL-23p19, respectively. Thus, being directed to,binding to or reacting with includes that the lipocalin muteinspecifically binds to IL-17A or IL-23p19, respectively. The term“specifically” in this context means that the lipocalin mutein reactswith an IL-17A protein or an IL-23p19 protein, as described herein, butessentially not with another protein. The term “another protein”includes any non-IL-17A or non-IL-23p19 protein, respectively, includingproteins closely related to or being homologous to IL-17A or IL-23p19against which the lipocalins disclosed herein are directed to. However,IL-17A or IL-23p19 proteins, fragments and/or variants from speciesother than human such as those described in the context of thedefinition “subject” are not excluded by the term “another protein”. Theterm “does not essentially bind” means that the lipocalin mutein of thepresent disclosure does not bind another protein, i.e., shows across-reactivity of less than 30%, preferably 20%, more preferably 10%,particularly preferably less than 9, 8, 7, 6 or 5%. Whether thelipocalin specifically reacts as defined herein above can easily betested, inter alia, by comparing the reaction of a lipocalin mutein ofthe present disclosure with IL-17A or IL-23p19 and the reaction of saidlipocalin with (an) other protein(s). “Specific binding” can also bedetermined, for example, in accordance with Western blots, ELISA-, RIA-,ECL-, IRMA-tests, FACS, IHC and peptide scans.

The amino acid sequence of a lipocalin mutein according to thedisclosure has a high sequence identity to respective lipocalin whencompared to sequence identities with another lipocalin (see also above).In this general context the amino acid sequence of a lipocalin mutein ofthe combination according to the disclosure is at least substantiallysimilar to the amino acid sequence of the corresponding lipocalin (thewild-type or reference lipocalin). A respective sequence of a lipocalinmutein of the combination according to the disclosure, beingsubstantially similar to the sequences of the corresponding lipocalin,has in some to the wild-type (or reference) lipocalin, one or more aminoacid embodiments at least 65%, at least 70%, at least 75%, at least 80%,at least 82%, at least 85%, at least 87%, at least 90% identity,including at least 95% identity to the sequence of the correspondinglipocalin. In this regard, a lipocalin mutein of the disclosure ofcourse may contain, in comparison substitutions as described hereinwhich renders the lipocalin mutein capable of binding to IL-17A orIL-23p19, respectively. Typically a mutein of a lipocalin includes oneor more mutations—relative to the native sequence lipocalin—of aminoacids in the four loops at the open end of the ligand binding site ofthe lipocalin (cf. above). As explained above, these regions areessential in determining the binding specificity of a lipocalin muteinfor a desired target. As an illustrative example, a mutein derived froma polypeptide of tear lipocalin, NGAL lipocalin or a homologue thereof,may have one, two, three, four or more mutated amino acid residues atany sequence position in the N-terminal region and/or in the threepeptide loops BC, DE, and FG arranged at the end of the β-barrelstructure that is located opposite to the natural lipocalin bindingpocket. As a further illustrative example, a mutein derived from apolypeptide of tear lipocalin or a homologue thereof, may have nomutated amino acid residues in peptide loop DE arranged at the end ofthe β-barrel structure, compared to wild type sequence of tearlipocalin.

A lipocalin mutein according to the disclosure includes one or more,such as two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen,nineteen or even twenty substitutions in comparison to the correspondingnative lipocalin, provided that such a lipocalin mutein should becapable of binding to IL-17A or IL-23p19, respectively. For example, alipocalin mutein can have a substitution at a position corresponding toa distinct position (i.e. at a corresponding position) of the wild-typelipocalin having the wild-type sequence of, for example, tear lipocalin,NGAL lipocalin, or any other lipocalin disclosed herein. In someembodiments a lipocalin mutein of the combination according to thedisclosure includes at least two amino acid substitutions, including 2,3, 4 or 5, sometimes even more, amino acid substitutions of a nativeamino acid by an arginine residue. Accordingly, the nucleic acid of aprotein ‘reference’ scaffold as described herein is subject tomutagenesis with the aim of generating a lipocalin mutein which iscapable of binding to IL-17A or IL-23p19, respectively.

Also, a lipocalin mutein of the present disclosure can comprise aheterologous amino acid sequence at its N- or C-Terminus, preferablyC-terminus, such as a Strep-tag, e.g., Strep II tag without affectingthe biological activity (binding to its target e.g. IL-17A or IL-23p19,respectively) of the lipocalin mutein. A preferred example of a tag isshown in SEQ ID NO: 17).

Likewise, a lipocalin mutein of the present disclosure may lack 1, 2, 3,4 or more amino acids at its N-terminal end and/or 1, 2 or more aminoacids at its C-terminal end, in comparison to the respective wild-typelipocalin; for example, SEQ ID NOs: 2-7 and 12-14.

Specifically, in order to determine whether an amino acid residue of theamino acid sequence of a lipocalin mutein different from a wild-typelipocalin corresponds to a certain position in the amino acid sequenceof a wild-type lipocalin, a skilled artisan can use means and methodswell-known in the art, e.g., alignments, either manually or by usingcomputer programs such as BLAST2.0, which stands for Basic LocalAlignment Search Tool or ClustalW or any other suitable program which issuitable to generate sequence alignments. Accordingly, a wild-typelipocalin can serve as “subject sequence” or “reference sequence”, whilethe amino acid sequence of a lipocalin different from the wild-typelipocalin described herein serves as “query sequence”. The terms“reference sequence” and “wild type sequence” are used interchangeablyherein.

In some embodiments a substitution (or replacement) is a conservativesubstitution. Nevertheless, any substitution—including non-conservativesubstitution or one or more from the exemplary substitutions listedbelow—is envisaged as long as the lipocalin mutein retains itscapability to bind to IL-17A or IL-23p19, respectively, and/or it has anidentity to the then substituted sequence in that it is at least 60%,such as at least 65%, at least 70%, at least 75%, at least 80%, at least85% or higher identical to the “original” sequence.

Conservative substitutions are generally the following substitutions,listed according to the amino acid to be mutated, each followed by oneor more replacement(s) that can be taken to be conservative: Ala→Gly,Ser, Val; Arg→Lys; Asn→Gln, His; Asp→Glu; Cys→Ser; Gln→Asn; Glu→Asp;Gly→Ala; His→Arg, Asn, Gln; Ile→Leu, Val; Leu→Ile, Val; Lys→Arg, Gln,Glu; Met→Leu, Tyr, Ile; Phe→Met, Leu, Tyr; Ser→Thr; Thr Ser; Trp→Tyr;Tyr→Trp, Phe; Val→Ile, Leu. Other substitutions are also permissible andcan be determined empirically or in accord with other known conservativeor non-conservative substitutions. As a further orientation, thefollowing eight groups each contain amino acids that can typically betaken to define conservative substitutions for one another:

a. Alanine (Ala), Glycine (Gly);b. Aspartic acid (Asp), Glutamic acid (Glu);c. Asparagine (Asn), Glutamine (Gln);d. Arginine (Arg), Lysine (Lys);e. Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Val);f. Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp);g. Serine (Ser), Threonine (Thr); andh. Cysteine (Cys), Methionine (Met)

If such substitutions result in a change in biological activity, thenmore substantial changes, such as the following, or as further describedbelow in reference to amino acid classes, may be introduced and theproducts screened for a desired characteristic. Examples of such moresubstantial changes are: Ala→Leu, lie; Arg→Gln; Asn→Asp, Lys, Arg, His;Asp→Asn; Cys→Ala; Gln→Giu; Giu→Gln; His→Lys; Ile→Met, Ala, Phe; Leu→Ala,Met, Norleucine; Lys→Asn; Met→Phe; Phe→Val, lie, Ala; Trp→Phe; Tyr→Thr,Ser; Val→Met, Phe, Ala.

Substantial modifications in the biological properties of the lipocalinare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties: (1) hydrophobic: norleucine, methionine, alanine, valine,leucine, iso-leucine; (2) neutral hydrophilic: cysteine, serine,threonine; (3) acidic: asparitic acid, glutamic acid; (4) basic:asparagine, glutamine, histidine, lysine, arginine; (5) residues thatinfluence chain orientation: glycine, proline; and (6) aromatic:tryptophan, tyrosine, phenylalanine.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Any cysteine residue not involved inmaintaining the proper conformation of the respective lipocalin also maybe substituted, generally with serine, to improve the oxidativestability of the molecule and prevent aberrant crosslinking. Conversely,cysteine bond (s) may be added to the lipocalin to improve itsstability.

Any mutation, including an insertion as discussed above, can beaccomplished very easily on the nucleic acid, e.g. DNA level usingestablished standard methods. Illustrative examples of alterations ofthe amino acid sequence are insertions or deletions as well as aminoacid substitutions. Such substitutions may be conservative, i.e. anamino acid residue is replaced with an amino acid residue of chemicallysimilar properties, in particular with regard to polarity as well assize. Examples of conservative substitutions are the replacements amongthe members of the following groups: 1) alanine, serine, and threonine;2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4)arginine and lysine; 5) iso-leucine, leucine, methionine, and valine;and 6) phenylalanine, tyrosine, and tryptophan. On the other hand, it isalso possible to introduce non-conservative alterations in the aminoacid sequence. In addition, instead of replacing single amino acidresidues, it is also possible to either insert or delete one or morecontinuous amino acids of the primary structure of tear lipocalin aslong as these deletions or insertion result in a stablefolded/functional mutein.

Modifications of the amino acid sequence include directed mutagenesis ofsingle amino acid positions in order to simplify sub-cloning of themutated lipocalin gene or its parts by incorporating cleavage sites forcertain restriction enzymes. In addition, these mutations can also beincorporated to further improve the affinity of a lipocalin mutein or afusion protein for a given target. Furthermore, mutations can beintroduced in order to modulate certain characteristics of the mutein orfusion protein such as to improve folding stability, serum stability,protein resistance or water solubility or to reduce aggregationtendency, if necessary. For example, naturally occurring cysteineresidues may be mutated to other amino acids to prevent disulphidebridge formation. It is also possible to deliberately mutate other aminoacid sequence position to cysteine in order to introduce new reactivegroups, for example for the conjugation to other compounds, such aspolyethylene glycol (PEG), hydroxyethyl starch (HES), biotin, peptidesor proteins, or for the formation of non-naturally occurring disulphidelinkages. The generated thiol moiety may be used to PEGylate or HESylatethe mutein or the fusion protein, for example, in order to increase theserum half-life of a respective lipocalin mutein or fusion protein.

In some embodiments, if one of the above moieties is conjugated to alipocalin mutein or a fusion protein of the disclosure, conjugation toan amino acid side chain can be advantageous. Suitable amino acid sidechains may occur naturally in the amino acid sequence of a humanlipocalin or may be introduced by mutagenesis. In case a suitablebinding site is introduced via mutagenesis, one possibility is thereplacement of an amino acid at the appropriate position by a cysteineresidue.

For example, such mutation includes at least one of Thr 40→Cys, Glu73→Cys, Arg 90→Cys, Asp 95→Cys or Glu 131→Cys substitution in the wildtype sequence of human tear lipocalin. The newly created cysteineresidue at any of these positions can in the following be utilized toconjugate the mutein or the fusion protein to a moiety prolonging theserum half-life of the mutein or a fusion protein thereof, such as PEGor an activated derivative thereof.

With respect to a mutein of human Lipocalin 2, exemplary possibilitiesof such a mutation to introduce a cysteine residue into the amino acidsequence of a lipocalin including human Lipocalin 2 mutein to includethe introduction of a cysteine (Cys) residue at at least at one of thesequence positions that correspond to sequence positions 14, 21, 60, 84,88, 116, 141, 145, 143, 146 or 158 of the wild type sequence of humanNGAL. In some embodiments where a human Lipocalin 2 mutein of thedisclosure has a sequence in which, in comparison to the sequence of theSWISS-PROT/UniProt Data Bank Accession Number P80188, a cysteine hasbeen replaced by another amino acid residue, the corresponding cysteinemay be reintroduced into the sequence. As an illustrative example, acysteine residue at amino acid position 87 may be introduced in such acase by reverting to a cysteine as originally present in the sequence ofSWISS-PROT accession No P80188. The generated thiol moiety at the sideof any of the amino acid positions 14, 21, 60, 84, 88, 116, 141, 145,143, 146 and/or 158 may be used to PEGylate or HESylate the mutein, forexample, in order to increase the serum half-life of a respective humanLipocalin 2 mutein or a fusion protein thereof.

In another embodiment, in order to provide suitable amino acid sidechains for conjugating one of the above moieties to a lipocalin muteinor a fusion protein according to the present disclosure, artificialamino acids may be introduced by mutagenesis. Generally, such artificialamino acids are designed to be more reactive and thus to facilitate theconjugation to the desired compound. One example of such an artificialamino acid that may be introduced via an artificial tRNA ispara-acetyl-phenylalanine.

For several applications of the muteins or fusion proteins disclosedherein it may be advantageous to use them in the form of conjugates, forexample, as fused to a moiety which is a protein, or a protein domain ora peptide. In some embodiments, a lipocalin mutein or a fusion proteinthereof is fused at the N-terminus or the C-terminus of the lipocalinmutein (including as comprised in a fusion protein of the disclosure) toa protein, a protein domain or a peptide, for instance, a signalsequence and/or an affinity tag.

Affinity tags such as the Strep-tag® or Strep-tag® II (Schmidt, T. G. M.et al. (1996) J. Mol. Biol. 255, 753-766), the myc-tag, the FLAG-tag,the His₆-tag or the HA-tag or proteins such as glutathione-S-transferasealso allow easy detection and/or purification of recombinant proteinsare further examples of suitable fusion partners. Finally, proteins withchromogenic or fluorescent properties such as the green fluorescentprotein (GFP) or the yellow fluorescent protein (YFP) are suitablefusion partners for lipocalin muteins of the disclosure as well.

In general, it is possible to label the lipocalin muteins or fusionproteins of the disclosure with a compound including any appropriatechemical substance or enzyme, which directly or indirectly generates adetectable compound or signal in a chemical, physical, optical, orenzymatic reaction. An example for a physical reaction and at the sametime optical reaction/marker is the emission of fluorescence uponirradiation or the emission of X-rays when using a radioactive label.Alkaline phosphatase, horseradish peroxidase and β-galactosidase areexamples of enzyme labels (and at the same time optical labels) whichcatalyze the formation of chromogenic reaction products. In general, alllabels commonly used for antibodies (except those exclusively used withthe sugar moiety in the Fc part of immunoglobulins) can also be used forconjugation to the lipocalin muteins or fusion proteins of thedisclosure. The lipocalin muteins or fusion proteins of the disclosuremay also be conjugated with any suitable therapeutically active agent,e.g., for the targeted delivery of such agents to a given cell, tissueor organ or for the selective targeting of cells, e.g., of tumor cellswithout affecting the surrounding normal cells. Examples of suchtherapeutically active agents include radionuclides, toxins, smallorganic molecules, and therapeutic peptides (such as peptides acting asagonists/antagonists of a cell surface receptor or peptides competingfor a protein binding site on a given cellular target). The lipocalinmuteins or fusion proteins of the disclosure may, however, also beconjugated with therapeutically active nucleic acids such as antisensenucleic acid molecules, small interfering RNAs, micro RNAs or ribozymes.Such conjugates can be produced by methods well known in the art.

As indicated above, a lipocalin mutein or a fusion protein of thedisclosure may in some embodiments be conjugated to a moiety thatextends the serum half-life of the mutein or the fusion protein (in thisregard see also PCT publication WO 2006/56464 where such conjugationstrategies are described with references to muteins of human neutrophilegelatinase-associated lipocalin with binding affinity for CTLA-4). Themoiety that extends the serum half-life may be a polyalkylene glycolmolecule, hydroxyethyl starch, fatty acid molecules, such as palmiticacid (Vajo & Duckworth 2000, Pharmacol. Rev. 52, 1-9), an Fc part of animmunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of animmunoglobulin, an albumin binding domain, an albumin binding peptide,or an albumin binding protein, transferrin to name only a few. Thealbumin binding protein may be a bacterial albumin binding protein, anantibody, an antibody fragment including domain antibodies (see U.S.Pat. No. 6,696,245, for example), or a lipocalin mutein with bindingactivity for albumin. Accordingly, suitable conjugation partner forextending the half-life of a lipocalin mutein or a fusion protein of thedisclosure includes a bacterial albumin binding domain, such as the oneof streptococcal protein G (König, T., & Skerra, A. (1998) J. Immunol.Methods 218, 73-83) or the one as shown in SEQ ID NO: 39. In addition,examples of albumin binding peptides that can be used as conjugationpartner are, for instance, those having a Cys-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cysconsensus sequence, wherein Xaa₁ is Asp, Asn, Ser, Thr, or Trp; Xaa₂ isAsn, Gln, His, lie, Leu, or Lys; Xaa₃ is Ala, Asp, Phe, Trp, or Tyr; andXaa₄ is Asp, Gly, Leu, Phe, Ser, or Thr as described in US patentapplication 2003/0069395 or Dennis et al. (Dennis, M. S., Zhang, M.,Meng, Y. G., Kadkhodayan, M., Kirchhofer, D., Combs, D. & Damico, L. A.(2002) J Biol Chem 277, 35035-35043).

In other embodiments, albumin itself (Osborn, B. L. et al., 2002, J.Pharmacol. Exp. Ther. 303, 540-548), or a biological active fragment ofalbumin can be used as conjugation partner of a lipocalin mutein or afusion protein of the disclosure. The term “albumin” includes all mammalalbumins such as human serum albumin or bovine serum albumin or ratalbumine

If the albumin-binding protein is an antibody fragment it may be adomain antibody. Domain Antibodies (dAbs) are engineered to allowprecise control over biophysical properties and in vivo half-life tocreate the optimal safety and efficacy product profile. DomainAntibodies are for example commercially available from Domantis Ltd.(Cambridge, UK and MA, USA).

When transferrin is used as a moiety to extend the serum half-life ofthe lipocalin muteins or the fusion proteins of the disclosure, themuteins or the fusion proteins can be genetically fused to the N or Cterminus, or both, of non-glycosylated transferrin. Non-glycosylatedtransferrin has a half-life of 14-17 days, and a transferrin-conjugatedmutein or fusion protein will similarly have an extended half-life. Thetransferrin carrier also provides high bioavailability, biodistributionand circulating stability. This technology is commercially availablefrom BioRexis (BioRexis Pharmaceutical Corporation, PA, USA).Recombinant human transferrin (DeltaFerrin™) for use as a proteinstabilizer/half-life extension partner is also commercially availablefrom Novozymes Delta Ltd. (Nottingham, UK).

If an Fc part of an immunoglobulin is used for the purpose to prolongthe serum half-life of the lipocalin muteins or fusion proteins of thedisclosure, the SynFusion™ technology, commercially available fromSyntonix Pharmaceuticals, Inc (MA, USA), may be used. The use of thisFc-fusion technology allows the creation of longer-actingbiopharmaceuticals and may, for example, consist of two copies of amutein linked to the Fc region of an antibody to improvepharmacokinetics, solubility, and production efficiency.

Yet another alternative to prolong the half-life of the lipocalinmuteins or fusion proteins of the disclosure is to fuse to the N- orC-terminus of the muteins (including as comprised in fusion proteins ofthe disclosure) long, unstructured, flexible glycine-rich sequences (forexample, poly-glycine with about 20 to 80 consecutive glycine residues).This approach disclosed in WO2007/038619, for example, has also beenterm “rPEG” (recombinant PEG).

If polyalkylene glycol is used as conjugation partner, the polyalkyleneglycol can be substituted, unsubstituted, linear or branched. It canalso be an activated polyalkylene derivative. Examples of suitablecompounds are polyethylene glycol (PEG) molecules as described in WO99/64016, in U.S. Pat. No. 6,177,074 or in U.S. Pat. No. 6,403,564 inrelation to interferon, or as described for other proteins such asPEG-modified asparaginase, PEG-adenosine deaminase (PEG-ADA) orPEG-superoxide dismutase (see for example, Fuertges et al. (1990) TheClinical Efficacy of Poly(Ethylene Glycol)-Modified Proteins J. Control.Release 11, 139-148). The molecular weight of such a polymer, such aspolyethylene glycol, may range from about 300 to about 70.000 Dalton,including, for example, polyethylene glycol with a molecular weight ofabout 10.000, of about 20.000, of about 30.000 or of about 40.000Dalton. Moreover, as e.g. described in U.S. Pat. No. 6,500,930 or6,620,413, carbohydrate oligo- and polymers such as starch orhydroxyethyl starch (HES) can be conjugated to a mutein or a fusionprotein of the disclosure for the purpose of serum half-life extension.

In addition, a lipocalin mutein or fusion protein disclosed herein maybe conjugated to a moiety that may confer new characteristics to thelipocalin muteins or fusion proteins of the disclosure such as enzymaticactivity or binding affinity for other molecules. Examples of suitablemoieties include alkaline phosphatase, horseradish peroxidase,gluthation-S-transferase, the albumin-binding domain of protein G,protein A, antibody fragments, oligomerization domains or toxins.

In addition, it may be possible to fuse a lipocalin mutein or fusionprotein disclosed herein with a separate enzyme active site such thatboth “components” of the resulting fusion protein together act on agiven therapeutic target. For example, the binding domain of thelipocalin mutein (including as comprised in a fusion protein of thedisclosure) may attach to the disease-causing target, allowing theenzyme domain to abolish the biological function of the target.

In some embodiments, a lipocalin mutein or a fusion protein of thedisclosed may be conjugated to a moiety via a linker (e.g. a peptidebond) that covalently links a lipocalin mutein of the disclosure andanother disclosed moiety to each other. This can be achieved, forexample, by expression of the linked lipocalin muteins as a singlepolypeptide connected by a peptide linker. A suitable peptide linker canbe comprised of a stretch of amino acids of arbitrary length containingany amino acids. A preferred linker design utilizes a repeated stretchof amino acids of glycines and serines following the formula (GxSy)n,where x is the number of glycine repeats and y the number of serinerepeats in a building block that is repeated n times. The values of eachof the variables x, y, and n can range from 0 to 100, preferably from 0to 10. Non-limiting examples are hereby provided with SEQ ID NO: 18 andSEQ ID NOs: 36-38.

In some other embodiments, chemical methods of covalently linking may beapplied to link a lipocalin mutein of the disclosure to anotherdisclosed moiety. One example is the use of bifunctional linkers thatallow reactive chemistry between the linker and an amino acid sidechain, for example, between a maleimide and and a free cysteine in alipocalin mutein, or an activated carboxylic acid ester and a primaryamine in the lipocalin mutein. This includes reaction with non-naturalamino acid side chains that may be included during protein expression,and which provide a functionality that can be selectively derivatised.In some still further embodiments, “click” chemistry, such as thecycloaddtion of an azide and an alkine, may be used to link one or moresubunits of a fusion protein of the disclosure.

The present disclosure also relates to nucleic acid molecules (DNA andRNA) that include nucleotide sequences encoding the lipocalin muteinsand the fusion proteins of the disclosure. Since the degeneracy of thegenetic code permits substitutions of certain codons by other codonsspecifying the same amino acid, the disclosure is not limited to aspecific nucleic acid molecule encoding a lipocalin mutein or a fusionprotein as described herein but encompasses all nucleic acid moleculesthat include nucleotide sequences encoding a functional mutein or afunctional fusion protein. In this regard, the present disclosureprovides nucleotide sequences encoding some exemplary lipocalin muteins,some exemplary fusion proteins generic as shown in SEQ ID NOs: 23-35,45-49 and 54.

In one embodiment of the disclosure, the method includes subjecting thenucleic acid molecule to mutagenesis at nucleotide triplets coding forat least one, sometimes even more, of the sequence positionscorresponding to the sequence positions 28, 36, 40-41, 49, 52, 68, 70,72-73, 75, 77, 79, 81, 87, 96, 100, 103, 106, 125, 127, 132 and 134 ofthe linear polypeptide sequence of human NGAL (SEQ ID NO: 8).

In another embodiment of the method according to the disclosure, anucleic acid molecule encoding a human tear lipocalin is firstlysubjected to mutagenesis at one or more of the amino acid sequencepositions 26-34, 55-58, 60-61, 64, 104-108 of the linear polypeptidesequence of human tear lipocalin (SEQ ID NO: 1). Secondly, the nucleicacid molecule encoding a human tear lipocalin is also subjected tomutagenesis at one or more of the amino acid sequence positions 101,111, 114 and 153 of the linear polypeptide sequence of the mature humantear lipocalin.

The disclosure also includes nucleic acid molecules encoding thelipocalin muteins and fusion proteins of the disclosure, which includeadditional mutations outside the indicated sequence positions ofexperimental mutagenesis. Such mutations are often tolerated or can evenprove to be advantageous, for example if they contribute to an improvedfolding efficiency, serum stability, thermal stability or ligand bindingaffinity of the muteins and the fusion proteins.

A nucleic acid molecule disclosed in this application may be “operablylinked” to a regulatory sequence (or regulatory sequences) to allowexpression of this nucleic acid molecule.

A nucleic acid molecule, such as DNA, is referred to as “capable ofexpressing a nucleic acid molecule” or capable “to allow expression of anucleotide sequence” if it includes sequence elements which containinformation regarding to transcriptional and/or translationalregulation, and such sequences are “operably linked” to the nucleotidesequence encoding the polypeptide. An operable linkage is a linkage inwhich the regulatory sequence elements and the sequence to be expressedare connected in a way that enables gene expression. The precise natureof the regulatory regions necessary for gene expression may vary amongspecies, but in general these regions include a promoter which, inprokaryotes, contains both the promoter per se, i.e. DNA elementsdirecting the initiation of transcription, as well as DNA elementswhich, when transcribed into RNA, will signal the initiation oftranslation. Such promoter regions normally include 5′ non-codingsequences involved in initiation of transcription and translation, suchas the −35/−10 boxes and the Shine-Dalgarno element in prokaryotes orthe TATA box, CAAT sequences, and 5′-capping elements in eukaryotes.These regions can also include enhancer or repressor elements as well astranslated signal and leader sequences for targeting the nativepolypeptide to a specific compartment of a host cell.

In addition, the 3′ non-coding sequences may contain regulatory elementsinvolved in transcriptional termination, polyadenylation or the like.If, however, these termination sequences are not satisfactory functionalin a particular host cell, then they may be substituted with signalsfunctional in that cell.

Therefore, a nucleic acid molecule of the disclosure can include aregulatory sequence, such as a promoter sequence. In some embodiments anucleic acid molecule of the disclosure includes a promoter sequence anda transcriptional termination sequence. Suitable prokaryotic promotersare, for example, the tet promoter, the lacUV5 promoter or the T7promoter. Examples of promoters useful for expression in eukaryoticcells are the SV40 promoter or the CMV promoter.

The nucleic acid molecules of the disclosure can also be part of avector or any other kind of cloning vehicle, such as a plasmid, aphagemid, a phage, a baculovirus, a cosmid or an artificial chromosome.

In one embodiment, the nucleic acid molecule is included in a phasmid. Aphasmid vector denotes a vector encoding the intergenic region of atemperent phage, such as M13 or f1, or a functional part thereof fusedto the cDNA of interest. After superinfection of the bacterial hostcells with such an phagemid vector and an appropriate helper phage (e.g.M13K07, VCS-M13 or R408) intact phage particles are produced, therebyenabling physical coupling of the encoded heterologous cDNA to itscorresponding polypeptide displayed on the phage surface (see e.g.Lowman, H. B. (1997) Annu. Rev. Biophys. Biomol. Struct. 26, 401-424, orRodi, D. J., and Makowski, L. (1999) Curr. Opin. Biotechnol. 10, 87-93).

Such cloning vehicles can include, aside from the regulatory sequencesdescribed above and a nucleic acid sequence encoding a lipocalin muteinor a fusion protein as described herein, replication and controlsequences derived from a species compatible with the host cell that isused for expression as well as selection markers conferring a selectablephenotype on transformed or transfected cells. Large numbers of suitablecloning vectors are known in the art, and are commercially available.

The DNA molecule encoding a lipocalin mutein or a fusion protein asdescribed herein, and in particular a cloning vector containing thecoding sequence of such a mutein can be transformed into a host cellcapable of expressing the gene. Transformation can be performed usingstandard techniques. Thus, the disclosure is also directed to a hostcell containing a nucleic acid molecule as disclosed herein.

The transformed host cells are cultured under conditions suitable forexpression of the nucleotide sequence encoding a lipocalin mutein or afusion protein of the disclosure. Suitable host cells can beprokaryotic, such as Escherichia coli (E. coli) or Bacillus subtilis, oreukaryotic, such as Saccharomyces cerevisiae, Pichia pastoris, SF9 orHigh5 insect cells, immortalized mammalian cell lines (e.g., HeLa cellsor CHO cells) or primary mammalian cells.

The disclosure also relates to a method for the production of apolypeptide as described herein, wherein the lipocalin mutein or thefusion protein is produced starting from the nucleic acid coding for thelipocalin mutein or the fusion protein by means of genetic engineeringmethods. The method can be carried out in vivo, the lipocalin mutein orthe fusion protein can for example be produced in a bacterial oreucaryotic host organism and then isolated from this host organism orits culture. It is also possible to produce a protein in vitro, forexample by use of an in vitro translation system.

When producing the lipocalin mutein, the fusion protein or the fragmentin vivo, a nucleic acid encoding such mutein is introduced into asuitable bacterial or eukaryotic host organism by means of recombinantDNA technology (as already outlined above). For this purpose, the hostcell is first transformed with a cloning vector that includes a nucleicacid molecule encoding a lipocalin mutein or a fusion protein asdescribed herein using established standard methods. The host cell isthen cultured under conditions, which allow expression of theheterologous DNA and thus the synthesis of the correspondingpolypeptide. Subsequently, the polypeptide is recovered either from thecell or from the cultivation medium.

In some embodiments, a nucleic acid molecule, such as DNA, disclosed inthis application may be “operably linked” to another nucleic acidmolecule of the disclosure to allow expression of a fusion protein ofthe disclosure. In this regard, an operable linkage is a linkage inwhich the sequence elements of the first nucleic acid molecule and thesequence elements of the second nucleic acid molecule are connected in away that enables expression of the fusion protein as a singlepolypeptide.

In addition, in some embodiments, the naturally occurring disulfide bondbetween Cys 76 and Cys 175 may be removed in NGAL muteins of thedisclosure (including as comprised in fusion proteins of thedisclosure). In some embodiments for Tlc muteins of the disclosure aswell (including as comprised in fusion proteins of the disclosure), thenaturally occurring disulfide bond between Cys 61 and Cys 153 may beremoved. Accordingly, such muteins or fusion proteins can be produced ina cell compartment having a reducing redox milieu, for example, in thecytoplasma of Gram-negative bacteria.

In case a lipocalin mutein or a fusion protein of the disclosureincludes intramolecular disulfide bonds, it may be preferred to directthe nascent polypeptide to a cell compartment having an oxidizing redoxmilieu using an appropriate signal sequence. Such an oxidizingenvironment may be provided by the periplasm of Gram-negative bacteriasuch as E. coli, in the extracellular milieu of Gram-positive bacteriaor in the lumen of the endoplasmatic reticulum of eukaryotic cells andusually favors the formation of structural disulfide bonds.

It is, however, also possible to produce a lipocalin mutein or a fusionprotein of the disclosure in the cytosol of a host cell, preferably E.coli. In this case, the polypeptide can either be directly obtained in asoluble and folded state or recovered in form of inclusion bodies,followed by renaturation in vitro. A further option is the use ofspecific host strains having an oxidizing intracellular milieu, whichmay thus allow the formation of disulfide bonds in the cytosol (Venturiet al. (2002) J. Mol. Biol. 315, 1-8.).

However, a lipocalin mutein or a fusion protein as described herein maynot necessarily be generated or produced only by use of geneticengineering. Rather, such a lipocalin mutein or a fusion protein canalso be obtained by chemical synthesis such as Merrifield solid phasepolypeptide synthesis or by in vitro transcription and translation. Itis for example possible that promising mutations are identified usingmolecular modeling and then to synthesize the wanted (designed)polypeptide in vitro and investigate the binding activity for IL-17A.Methods for the solid phase and/or solution phase synthesis of proteinsare well known in the art (see e.g. Bruckdorfer, T. et al. (2004) Curr.Pharm. Biotechnol. 5, 29-43).

In another embodiment, the lipocalin muteins or the fusion proteins ofthe disclosure may be produced by in vitro transcription/translationemploying well-established methods known to those skilled in the art.

The skilled worker will appreciate methods useful to prepare lipocalinmuteins contemplated by the present disclosure but whose protein ornucleic acid sequences are not explicitly disclosed herein. As anoverview, such modifications of the amino acid sequence include, e.g.,directed mutagenesis of single amino acid positions in order to simplifysub-cloning of a mutated lipocalin gene or its parts by incorporatingcleavage sites for certain restriction enzymes. In addition, thesemutations can also be incorporated to further improve the affinity of alipocalin mutein or a fusion protein for its target (e.g. IL-17A orIL-23p19, respectively). Furthermore, mutations can be introduced tomodulate certain characteristics of the mutein or the fusion protein,such as, to improve folding stability, serum stability, proteinresistance or water solubility or to reduce aggregation tendency, ifnecessary. For example, naturally occurring cysteine residues may bemutated to other amino acids to prevent disulphide bridge formation.

The lipocalin muteins and fusion proteins, disclosed herein, as well astheir derivatives can be used in many fields similar to antibodies orfragments thereof. For example, the lipocalin muteins and/or fusionproteins, as well as their respective derivatives, can be used forlabeling with an enzyme, an antibody, a radioactive substance or anyother group having biochemical activity or defined bindingcharacteristics. By doing so, their respective targets thereof can bedetected or brought in contact with them. In addition, lipocalin muteinsand/or fusion proteins of the disclosure can serve to detect chemicalstructures by means of established analytical methods (e.g., ELISA orWestern Blot) or by microscopy or immunosensorics. In this regard, thedetection signal can either be generated directly by use of a suitablemutein, or a suitable fusion protein; or indirectly by immunochemicaldetection of the bound mutein via an antibody.

Other protein scaffolds that can be engineered in accordance with thepresent invention to provide protein muteins that bind IL-17 and/orIL-23 with detectable affinity include: an EGF-like domain, aKringle-domain, a fibronectin type I domain, a fibronectin type IIdomain, a fibronectin type III domain, a PAN domain, a G1a domain, aSRCR domain, a Kunitz/Bovine pancreatic trypsin Inhibitor domain,tendamistat, a Kazal-type serine protease inhibitor domain, a Trefoil(P-type) domain, a von Willebrand factor type C domain, anAnaphylatoxin-like domain, a CUB domain, a thyroglobulin type I repeat,LDL-receptor class A domain, a Sushi domain, a Link domain, aThrombospondin type I domain, an immunoglobulin domain or a animmunoglobulin-like domain (for example, domain antibodies or camelheavy chain antibodies), a C-type lectin domain, a MAM domain, a vonWillebrand factor type A domain, a Somatomedin B domain, a WAP-type fourdisulfide core domain, a F5/8 type C domain, a Hemopexin domain, an SH2domain, an SH3 domain, a Laminin-type EGF-like domain, a C2 domain,“Kappabodies” (III. et al. “Design and construction of a hybridimmunoglobulin domain with properties of both heavy and light chainvariable regions” Protein Eng 10:949-57 (1997)), “Minibodies” (Martin etal. “The affinity-selection of a minibody polypeptide inhibitor of humaninterleukin-6” EMBO J 13:5303-9 (1994)), “Diabodies” (Holliger et al.“‘Diabodies’: small bivalent and bispecific antibody fragments” PNAS USA90:6444-6448 (1993)), “Janusins” (Traunecker et al. “Bispecific singlechain molecules (Janusins) target cytotoxic lymphocytes on HIV infectedcells” EMBO J 10:3655-3659 (1991) and Traunecker et al. “Janusin: newmolecular design for bispecific reagents” Int J Cancer Suppl 7:51-52(1992), a nanobody, an adnectin, a tetranectin, a microbody, an affilin,an affibody an ankyrin, a crystallin, a knottin, ubiquitin, azinc-finger protein, an autofluorescent protein, an ankyrin or ankyrinrepeat protein or a leucine-rich repeat protein, an avimer (Silverman,Lu Q, Bakker A, To W, Duguay A, Alba B M, Smith R, Rivas A, Li P, Le H,Whitehorn E, Moore K W, Swimmer C, Perlroth V, Vogt M, Kolkman J,Stemmer W P 2005, Nat Biotech, December; 23(12):1556-61, E-Publicationin Nat Biotech. 2005 Nov. 20 edition); as well as multivalent avimerproteins evolved by exon shuffling of a family of human receptor domainsas also described in Silverman J, Lu Q, Bakker A, To W, Duguay A, Alba BM, Smith R, Rivas A, Li P, Le H, Whitehorn E, Moore K W, Swimmer C,Perlroth V, Vogt M, Kolkman J, Stemmer W P, Nat Biotech, December;23(12):1556-61, E-Publication in Nat. Biotechnology. 2005 Nov. 20edition).

Additional objects, advantages, and features of this disclosure willbecome apparent to those skilled in the art upon examination of thefollowing Examples and the attached Figures thereof, which are notintended to be limiting. Thus, it should be understood that although thepresent disclosure is specifically disclosed by exemplary embodimentsand optional features, modification and variation of the disclosuresembodied therein herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this disclosure.

V. EXAMPLES Example 1: Affinity of Lipocalin Mutein to IL-17A Measuredby SPR

To measure the binding affinity of the lipocalin mutein of SEQ ID NO: 1to IL-17A, a surface plasmon resonance (SPR) based assay was employedutilizing a Biacore T200 instrument (GE Healthcare). In the SPR affinityassay (FIG. 1), IL-17A was immobilized on a sensor chip using standardamine chemistry: The surface of the chip was activated using1-Ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) andN-hydroxysuccinimid (NHS). Subsequently, 5 μg/mL of IL-17A solution in10 mM Acetate pH 5 was applied at a flow rate of 10 μL/min until animmobilization level of 279 resonance units (RU) was achieved. Residualactivated groups were quenched with ethanolamine. The reference channelsunderwent blank immobilization by treatment with EDC/NHS followingethanolamine.

To determine the affinity, four dilutions of SEQ ID NO: 1 were preparedin HBS-EP+ buffer and applied to the prepared chip surface. The bindingassay was carried out with a contact time of 300 s, dissociation time of1200 s and applying a flow rate of 30 μL/min. All measurements wereperformed at 25° C. Regeneration of the immobilized IL-17A surface wasachieved with consecutive injections of 10 mM aqueous H₃PO₄ (30 s) and10 mM glycine-HCl pH 1.5 (15 s) followed by an extra wash with runningbuffer and a stabilization period of 30 s. Prior to the proteinmeasurements three startup cycles were performed for conditioningpurposes. Data were evaluated with Biacore T200 Evaluation software (V1.0). Double referencing was used. The 1:1 binding model was used to fitthe raw data.

The resulting fit curves are shown in FIG. 1. The data shows that SEQ IDNO: 1 bound with high affinity to IL-17A, with an association rateconstant of k_(a)=7.0×10⁴ M⁻¹sec⁻¹ and a dissociation rate constant ofk_(d)=5.3×10⁻⁵ sec⁻¹, resulting in a dissocation constant of K_(D)=0.8nM.

Example 2: Affinity of Lipocalin Mutein to IL-17 A/F Measured by SPR inan Alternative Format

To measure the binding affinity of the lipocalin mutein of SEQ ID NO: 1to IL-17AF in a Surface Plasmon Resonance (SPR) based assay in analternative assay format—capturing SEQ ID NO: 1 as a ligand and applyinghuman IL-17 A/F as an analyte—a Biacore T200 instrument (GE Healthcare)was used. In this format, human IL-17 A/F was employed (as opposed tothe homodimeric IL-17A, which was used in Example 1) to avoid potentialavidity effects in the assay. SEQ ID NO: 1 was biotinylated for 2 h atroom temperature applying an appropriate excess of EZ-LinkNHS-PEG4-Biotin (Thermo, Cat #21329) followed by separation ofnon-reacted Biotin using a Zeba Spin Desalting Plate (Thermo, Cat#21329) according to the manufacturers instructions.

In the SPR affinity assay, biotinylated SEQ ID NO: 1 was captured on asensor chip CAP using the Biotin CAPture Kit (GE Healthcare): The sensorChip CAP was pre-immobilized with an ssDNA oligonucleotide. UndilutedBiotin CAPture Reagent (streptavidin conjugated with the complementaryss-DNA oligonucleotide) was applied at a flow rate of 2 μL/min for 300s. Subsequently, 0.4 to 10 μg/mL of biotinylated SEQ ID NO: 1 wasapplied for 300 s at a flow rate of 5 μL/min. The reference channel wasloaded with Biotin CAPture Reagent only.

To determine the binding affinity, five dilutions of human IL-17 A/F(0.2-20 nM) were prepared in HBS-EP+ buffer and applied to the preparedchip surface. Applying a flow rate of 30 μL/min, a single cycle kineticsapproach was used with a sample contact time of 300 s and a dissociationtime of 2400 s. After ligand immobilization, all 5 concentrations wereapplied consecutively in ascending order before the dissociation wasmonitored. All measurements were performed at 25° C. Regeneration of theSensor Chip CAP surface was achieved with an injection of 6 M Gua-HClwith 0.25 M NaOH followed by an extra wash with running buffer and astabilization period of 120 s. Data were evaluated with Biacore T200Evaluation software (V 1.0). Double referencing was used. A single cyclekinetic 1:1 binding model was used to fit the raw data.

The resulting fit curves are shown in FIG. 2. The data shows that SEQ IDNO: 1 bound with high affinity to human IL-17 A/F, with an associationrate constant of k_(a)=1.2×10⁶ M⁻¹sec⁻¹ and a dissociation rate constantof k_(d)=1.2×10⁻⁴sec⁻¹, resulting in a calculated equilibriumdissocation constant of K_(D)=100 pM. A comparison to Example 1 showsthat the result of the SPR assay is somewhat dependent on the assayformat. However, in both formats, the affinity of SEQ ID NO: 1 to humanIL-17A and human IL-17 A/F, respectively, is high and in thesubnanomolar range. The assay is important for allowing a comparisonbetween SEQ ID NO: 1 and fusions containing this mutein (see belowExample 11).

Example 3: Competitive Mode of Action of Lipocalin Mutein Binding toIL-17A

Whether SEQ ID NO: 1 binds to IL-17A in a competitive mode was tested invitro using a competition ELISA format (FIG. 3). In this experiment, aconstant concentration of biotinylated human IL-17A was incubated withvariable concentrations of SEQ ID NO: 1 for 1 h. After thispre-incubation in solution, an aliquot of the lipocalin mutein/IL-17Amixture was transferred to an ELISA plate coated with human IL-17RA tomeasure the concentration of hIL-17A that was not blocked to bind tohIL-17RA (FIG. 3).

All incubation steps were performed with shaking at 300 rpm, and theplate was washed after each incubation step with 80 μl PBS-T buffer(PBS, 0.05% Tween 20) for five times using a Biotek EL405 select CWwasher. In the first step, a 384 well MSD plate was directly coated with20 μl of soluble human IL-17RA at a concentration of 1 μg/ml in PBS overnight at 4° C. After washing, the receptor coated wells were blockedwith 60 μl PBS-T/BSA (2% BSA in PBS containing 0.1% Tween 20) for 1 h atroom temperature.

A fixed concentration of 0.01 nM human IL-17A was incubated in solutionwith varying concentrations of SEQ ID NO: 1, or with SEQ ID NO: 41 as anegative control, using a suitable starting concentration of SEQ ID NO:1 and SEQ ID NO: 41 which was serially diluted at a 1:4 ratio down tothe picomolar range in PBS-T/BSA buffer. After 1h incubation at roomtemperature, 20 μl of the reaction mixture was transferred to thehIL-17RA-coated MSD plate to capture unbound (free) or non-competitivelybound hIL-17A for 20 min at RT. To allow for transformation of ELISAreadout results into absolute free hIL-17A concentrations (cf. below), astandard curve containing varying concentrations of hIL-17A was preparedin PBS-T/BSA and incubated for 20 min on the same MSD plate as well.

To allow for detection and quantitation of bound biotinylated hIL-17A,the residual supernatants were discarded and 20 μl Strepavidin Sulfo-tagwas added at a concentration of 1 μg/mL in PBS-T/BSA and incubated for 1h at RT. After washing, 60 μl MSD Read Buffer T (2×) was added to eachwell and the plate was read within 15 min.

The resulting Electrochemoluminescence (ECL) signal was measured usingthe SECTOR Imager 2400 (Meso Scale Discovery). The evaluation wasperformed as follows: free IL-17A concentration c(IL-17A)_(free) wascalculated (from the standard curve determined in parallel) and plottedversus SEQ ID NO: 1 concentration, c(SEQ ID NO: 1). To obtain the SEQ IDNO: 1 concentration at which formation of the IL-17A/IL-17 RA-complexwas blocked by 50% (IC50), the curves were fitted by nonlinearregression with a single-sites binding model according toc(IL-17A)_(free)=c(IL-17A)_(tot)/(1+c(SEQ ID NO: 1)/IC50)), with thetotal tracer concentration c(IL-17A)_(tot) and the IC50 value as freeparameters. Curve fitting was performed using GraphPad Prism 4 software.

FIG. 3 shows that the negative control SEQ ID NO: 41 does not bind tohIL-17A; in contrast, SEQ ID NO: 1 demonstrates a strong competitivebinding to hIL-17A, with a fitted IC50 value of 75 pM.

Example 4: Specificity and Species Crossreactivity of Lipocalin Muteinto IL-17A

Specificity and species crossreactivity (FIG. 4) of the lipocalin muteinof SEQ ID NO: 1 were assayed by a “solution competition ELISA”, theprinciple of which was as follows: A constant concentration of SEQ IDNO: 1 was incubated with variable concentrations of ligands the ligandshuman IL-17A, cynomolgus monkey IL-17A (cIL-17A), and marmoset IL-17A or1 h. After this pre-incubation in solution, an aliquot of themutein/ligand mixture was transferred to an ELISA plate withbiotinylated hIL-17A immobilized via neutravidin to measure theremaining concentration of free SEQ ID NO: 1. The concentration of free(non ligand-bound) SEQ ID NO: 1 was determined via a quantitative ELISAsetup (FIG. 4). Note that this assay relied on that all ligands weretargeting the same binding site on the SEQ ID NO: 1, i.e. that theligands bound to the SEQ ID NO: 1 in competition with each other.

In the following detailed experimental protocol, incubation and washingsteps were performed as described above in the competition ELISAprotocol. A 384-well plate suitable for fluorescence measurements(Greiner FLUOTRAC™ 600, black flat bottom, high-binding) was coated with20 μl of Neutravidin at a concentration of 5 μg/ml in PBS over night at4° C. After washing, the Neutravidin-coated wells were blocked with 100μl blocking buffer (PBS-T/BSA) for 1h at room temperature. After washingagain, 20 μl biotinylated hIL-17A at a concentration of 1 μg/mL in PBSwas added for 1h at room temperature, and excess reagent was removed.

A fixed concentration of 0.25 nM SEQ ID NO: 1 was incubated in solutionwith varying concentrations of ligands (hIL-17A, cIL-17A and marmosetIL-17A), using a suitable starting concentration which was seriallydiluted at a 1:3 ratio down to the picomolar range in PBS-T/BSA. After1h incubation at room temperature, 20 μl of the reaction mixture wastransferred to the 384-well plate upon which biotinylated hIL-17A wasimmobilized to capture unbound (free) SEQ ID NO: 1 for 20 min at RT. Toallow for transformation of ELISA readout results into absolute free SEQID NO: 1 concentrations (cf. below), a standard curve containing varyingconcentrations of SEQ ID NO: 1 was prepared in PBS-T/BSA and incubatedfor 20 min on the same ELISA plate as well.

The residual supernatants were discarded and 20 μl HRP-labeledanti-lipocalin antibody was added at a predetermined optimalconcentration in PBS-T/BSA and incubated for 1 h at RT. Theanti-lipocalin antibody had been obtained by immunization of rabbitswith a mixture of lipocalin muteins, and was subsequently coupled to HRPusing a kit (EZ-link Plus Activated Peroxidase, Thermo Scientific)according to the manufacturer's instructions, to obtain the antibody-HRPconjugate. After washing, 20 μl fluorogenic HRP substrate (Quantablue,Pierce) was added to each well, and the reaction was allowed to proceedfor 60 minutes. The fluorescence intensity of every well on the platewas read using a Genios Plus Microplate reader (Tecan). To evaluate thedata, free SEQ ID NO: 1 concentration, c(SEQ ID NO: 1)_(free), wascalculated based on the standard curve results, and plotted versusligand concentration, c(Ligand). To obtain the ligand concentration atwhich formation of the IL-17A/SEQ ID NO: 1 complex was blocked by 50%(IC50), the curves were fitted by nonlinear regression with asingle-sites binding model according to c(SEQ ID NO: 1)_(free)=c(SEQ IDNO: 1)_(tot)/(1+c(Ligand)/IC50)), with the total tracer concentrationc(SEQ ID NO: 1)_(tot) and the IC50 value as free parameters. Curvefitting was performed using GraphPad Prism 4 software.

In summary, curve fitting yielded the following results:IC50_(hIL-17A)=0.1 nM, IC50_(cIL-17A)=0.1 nM andIC50_(marmoset IL-17A)=0.2 nM. As depicted in FIG. 4, the datademonstrates that SEQ ID NO: 1 displays high affinity towards IL-17Afrom human, cynomolgus monkey and marmoset monkey, and therefore, showsthat SEQ ID NO: 1 is fully crossreactive with cynomolgus and marmosetmonkey IL-17A.

Example 5: Lipocalin-Mutein-Mediated Blockade of IL-17A Induced G-CSFSecretion in a Cell-Based Assay

We employed a cell-based assay to demonstrate the ability of SEQ ID NO:1 to block the biological activity of IL-17A. The assay was based onIL-17A-induced secretion of G-CSF in U87-MG cells (ATCC catalog#HTB-14). This highly sensitive functional assay was employed to comparethe potency of SEQ ID NO: 1 to two benchmark antibodies that aredeveloped clinically. In this assay, recombinant hIL-17A waspreincubated with SEQ ID NO: 1, benchmark antibody molecules or controlsand added to the cells. Besides SEQ ID NO: 1, the following benchmarksand controls were included in the assay: benchmark antibody 1 (heavychain SEQ ID NO: 53; light chain SEQ ID NO: 54), benchmark antibody 2(heavy chain SEQ ID 55; light chain SEQ ID NO: 56) and SEQ ID NO: 2 anda human IgG isotype antibody (Dianova, CAT #009-000-002) as negativecontrols. The concentration of G-CSF in the supernatant was thenmeasured by ELISA.

U87-MG cells were cultured in cell culture flasks under standardconditions (Dulbecco's Modified Eagle Medium DMEM (PAN Biotech GmbH)containing 10% fetal calf serum FCS (PAA Laboratories), 37° C., 5% CO₂atmosphere).

On day 1 of the experiment, the adherent cells were dissociated fromtheir substrate with Accutase (PAA Laboratories) according to themanufacturer's instructions. Subsequently, cells were centrifuged downfor 5 minutes at 1000 rpm, resuspended in medium and filtered through a100 μm cell strainer (Falcon) to remove cell aggregates. Cells were thenseeded in 96-well flat bottom tissue culture plates (Greiner) at adensity of 5000 cells per well using an end volume of 100 μl. They wereincubated overnight under standard conditions.

SEQ ID NO: 1, SEQ ID NO: 2, a human IgG isotype antibody (Dianova, CAT#009-000-002), benchmark antibody 1 and benchmark antibody 2 (asdescribed above) were the molecules under study (“MUS”). On day 2, themedium of the cells grown in the 96-well plate was replaced by 80 μlfresh medium and a fixed concentration of 0.1 nM recombinant hIL-17A anda dilution series of MUS-solutions were subsequently added to the cells.This was done in triplicate for each MUS or control. The cells wereincubated for a further 20-24 hours under standard conditions. Beforecollection of the supernatants for measurement of G-CSF levels, wellswere visually inspected with a microscope. Wells that exhibitedconsiderable cell death or the presence of cellular aggregates wereexcluded from evaluation. G-CSF levels were determined using the G-CSFUltra-Sensitive Kit from MSD. To evaluate the data, the G-CSFconcentration in arbitrary units was plotted versus the MUSconcentration, c(MUS). To obtain the MUS concentration at whichinduction of G-CSF production by U-87 MG cells was reduced to 50%(IC50), the curves were fitted by nonlinear regression with asingle-sites binding model according toc(G-CSF)=c(G-CSF)_(Min)+[c(G-CSF)_(Max)−c(G-CSF)_(Min)]/[1+c(MUS)/IC50],with free parameters being IC50, the induced G-CSF concentrationc(G-CSF)_(Max), and the uninduced G-CSF concentration c(G-CSF)_(Min).Here, it was assumed that c(G-CSF)_(Max) and c(G-CSF)_(Min) wereindependent of the antagonist or control molecule under study, and theywere therefore fitted to common values for all molecules.

FIG. 5 shows a representative example of the experiment, which wasperformed in duplicate. The resulting average EC50 value for SEQ ID NO:1 was 0.13 nM (0.17 nM in the first experiment, 0.10 nM in the repeatexperiment), which was significantly more potent that benchmark 1, whichexhibited an EC50=2.33 (2.65/2.01) nM, and in a similar range comparedto benchmark 2, with an EC50=0.12 (0.14/0.10) nM. Negative controls,consisting of SEQ ID NO: 2 and a human IgG isotype antibody (Dianova,CAT #009-000-002, not shown in FIG. 5), had no effect on IL-17A-inducedG-CSF production of the cells.

Example 6: Affinity of Lipocalin Mutein to IL-23 Measured by SPR

To measure the binding affinity of the lipocalin muteins of SEQ ID NO: 2to human IL-23, a surface plasmon resonance (SPR) based assay wasemployed utilizing a Biacore T200 instrument (GE Healthcare). In the SPRaffinity assay (FIG. 6), hIL-23 was immobilized on a sensor chip usingstandard amine chemistry: The surface of the chip was activated usingEDC and NHS. Subsequently, 5 μg/mL of hIL-23 solution in 10 mM acetatepH 4.5 was applied at a flow rate of 10 μL/min until a sufficientimmobilization level was achieved. Residual activated groups werequenched with ethanolamine. The reference channels were treated withEDC/NHS following ethanolamine (blank immobilization).

To determine the binding affinity of SEQ ID NO: 2, five dilutions of SEQID NO: 2 were prepared in HBS-EP+ buffer and applied to the preparedchip surface. The binding assay was carried out with a contact time of300 s, dissociation time of 1200 s and applying a flow rate of 30μL/min. All measurements were performed at 25° C. Regeneration of theimmobilized hIL-23 surface was achieved by injection of 10 mM aqueousH₃PO₄ (30 s) followed by an extra wash with running buffer and astabilization period of 30 s. Prior to the protein measurements threestartup cycles were performed for conditioning purposes. Data wereevaluated with Biacore T200 Evaluation software (V 1.0). Doublereferencing was used. The 1:1 binding model was used to fit the rawdata.

FIG. 6 shows a representative example of the experiment, which wereperformed in triplicate. The resulting fit curves demonstrate that SEQID NO: 2 bound with high affinity to hIL-23, with an association rateconstant of k_(a)=5.0×10⁴ M⁻¹sec⁻¹ and a dissociation rate constant ofk_(d)=1.9×10⁻⁵ sec⁻¹. The average dissocation constant determined inthree replicate experiments amounted to K_(D)=0.35±0.20 nM,demonstrating the high-affinity interaction between SEQ ID NO: 2 andhuman IL-23.

Example 7: Affinity of Lipocalin Mutein to IL-23 Measured by SPR in anAlternative Format

To measure the binding affinity of SEQ ID NO: 2 to human IL-23 usingsurface plasmon resonance (SPR) in an alternative assay format—capturingSEQ ID NO: 2 as a ligand and applying hIL-23 as an analyte—a BiacoreT200 instrument (GE Healthcare) was used. SEQ ID NO: 2 and controls werebiotinylated as described in Example 2. In the SPR affinity assay,biotinylated SEQ ID NO: 2 was captured on a sensor chip CAP using theBiotin CAPture Kit (GE Healthcare). To this end, undiluted BiotinCAPture Reagent was applied at a flow rate of 2 μL/min for 300 s.Subsequently, 0.4 to 10 μg/mL of biotinylated SEQ ID NO: 2 or controlswas applied for 300 s at a flow rate of 5 μL/min. The reference channelwas loaded with Biotin CAPture Reagent only.

To determine the binding affinity, five dilutions of hIL-23 (7-600 nM)were prepared in HBS-EP+ buffer supplemented with 350 mM NaCl andapplied to the prepared chip surface. The addition of 350 mM NaCl wasrequired to minimize non-specific interaction of hIL-23 with the chipsurface. Applying a flow rate of 30 μL/min, a single cycle kineticsapproach was used with a sample contact time of 300 s and a dissociationtime of 4000 s or 7200 s. After ligand immobilization, all 5concentrations were applied consecutively in ascending order before thedissociation was monitored. All measurements were performed at 25° C.Regeneration of the Sensor Chip CAP surface and data evaluation wasaccomplished as described in Example 2.

The resulting fit curves are shown in FIG. 7. The data shows that SEQ IDNO: 2 bound with rather high affinity to hIL-23, with an associationrate constant of k_(a)=1.23×10⁻⁴ M⁻¹sec⁻¹ and a dissociation rateconstant of k_(d)=3.55×10⁻⁵ sec⁻¹, resulting in a calculated equilibriumdissocation constant of K_(D)=2.9 nM. A comparison to Example 6 shows astrong drop in affinity when using this SPR assay format, which isattributable to the high and nonphysiological concentration of NaCl (350mM) that had to be included in the buffer to minimize nonspecificinteractions of hIL-23 with the chip surface, and therefore facilitatecarrying out the assay in this format. The assay is important forallowing a comparison between SEQ ID NO: 2 and fusions containing thismutein (see below Example 11).

Example 8: Competitive Mode of Action of Lipocalin Mutein Binding toIL-23

Whether the lipocalin mutein SEQ ID NO: 2 binds to hIL-23 in acompetitive mode was tested in vitro using a competition ELISA format(FIG. 8), in analogy to Example 3, but using hIL-23 as the target.

All incubation steps were performed with shaking 300 rpm, and the platewas washed after each incubation step with 80 μl PBS/0.05% Tween 20 forfive times using a Biotek EL405 select CW washer. A 384 well MSD platewas directly coated with 20 μl of soluble human IL-23 receptor at aconcentration of 1 μg/ml in PBS over night at 4° C. After washing, thereceptor-coated wells were blocked with 60 μl PBS-T/BSA for 1 h at roomtemperature.

A fixed concentration of 0.01 nM biotinylated human IL-23 was incubatedin solution with varying concentrations of SEQ ID NO: 2, or with SEQ IDNO: 43 as a negative control, using suitable starting concentrationswhich were serially diluted at a 1:4 ratio down to the picomolar rangein PBS-T/BSA. After 1h incubation at room temperature, 20 μl of thereaction mixture was transferred to the IL-23 receptor-coated MSD plateto capture unbound (free) or non-competitively bound hIL-23 for 20 minat RT. To allow for transformation of ELISA readout results intoabsolute free hIL-23 concentrations (cf. below), a standard curvecontaining varying concentrations of hIL-23 starting with 100 nM (1:4serially diluted in 11 steps) was prepared in PBS-T/BSA and incubatedfor 20 min on the same MSD plate as well. To allow for detection andquantitation of bound biotinylated hIL-23, the residual supernatantswere discarded and 20 μl Strepavidin Sulfo-tag was added at aconcentration of 1 μg/mL in PBS-T/BSA and incubated for 1 h at RT. Afterwashing, 60 μl MSD Read Buffer T (2×) was added to each well and theplate was read within 15 min.

The resulting ECL signal was measured using the SECTOR Imager 2400 (MesoScale Discovery). The evaluation was performed in analogy to Example 3.As shown in FIG. 8, the negative control SEQ ID NO: 43 did not bind tohIL-23, in contrast, SEQ ID NO: 2 demonstrated a competitive binding tohIL23, with a fitted IC50 value of 0.54 nM.

Example 9: Specificity and Species Crossreactivity of Lipocalin Muteinto IL-23

The specificity and species crossreactivity of the lipocalin mutein ofSEQ ID NO: 2 (FIG. 9) was assayed by a “solution competition ELISA”, inanalogy to Example 4, but studying different ligands, namely, IL-23 fromhuman, cynomolgus monkey (cIL-23), marmoset monkey and mouse.

In the following detailed experimental protocol, incubation and washingsteps were performed as described above in the competition ELISAprotocol. A 384-well plate suitable for fluorescence measurements(Greiner FLUOTRAC™ 600, black flat bottom, high-binding) was coated with20 μl of Neutravidin at a concentration of 5 μg/ml in PBS over night at4° C. After washing, the Neutravidin-coated wells were blocked with 100μl PBS-T/BSA for 1h at room temperature. After washing again, 20 μlbiotinylated hIL-23-Bio at a concentration of 0.25 μg/mL in PBS wasadded for 1h at room temperature, and excess reagent was removed.

A fixed concentration of 0.5 nM SEQ ID NO: 2 was incubated in solutionwith varying concentrations of the ligands (hIL-23, cIL-23, marmosetIL-23 and mouse IL-23), using a suitable starting concentration whichwas serially diluted at a 1:3 ratio down to the picomolar range inPBS-T/BSA. After 20 minutes incubation at room temperature, 20 μl of thereaction mixture was transferred to the hIL-23 coated 384-well plate tocapture unbound (free) SEQ ID NO: 2 for 20 min at RT. To allow fortransformation of ELISA readout results into absolute free SEQ ID NO: 2concentrations (cf. below), a standard curve containing varyingconcentrations of SEQ ID NO: 2 was prepared in PBS-T/BSA and incubatedfor 20 min on the same MSD plate as well.

To quantitate plate-captured SEQ ID NO: 2, the residual supernatantswere discarded and 20 μl HRP-labeled anti-lipocalin antibody was addedat a predetermined optimal concentration in PBS-T/BSA and incubated for1 h at RT. The anti-ipocalin antibody had been obtained by immunizationof rabbits with a mixture of lipocalin muteins, and was subsequentlycoupled to HRP using a kit (EZ-link Plus Activated Peroxidase, ThermoScientific) according to the manufacturer's instructions, to obtain theantibody-HRP conjugate. Further handling of the plates, data acquisitionand evaluation were performed as described in Example 4.

As shown in FIG. 9, the result demonstrates that SEQ ID NO: 2 is fullycrossreactive with human and mouse IL-23, and displays a somewhatreduced affinity towards IL-23 of cynomolgus and marmoset monkey, withIC50_(hIL-23)=0.9 nM, IC50_(cIL-23)=4.8 nM, IC50_(marmIL-23)=12 nM andIC50_(mIL-23)=0.5 nM.

Example 10: Lipocalin-Mutein-Mediated Blockade of IL-23 in Cell-BasedProliferation Assays

The ability of the lipocalin mutein of SEQ ID NO: 2 to neutralize thebiological activity of hIL-23 was assessed by the application ofshort-term proliferation bioassays employing cells that recombinantlyexpress the human IL-23 receptor. The Ba/F3 transfectant cell lineexpresses both subunits of the receptor, hIL-23R and hIL-12Rβ1, and isresponsive to both human IL-23 and cynomolgus monkey IL-23. The Ba/F3cells proliferate responding to hIL-23 in a dose-dependent manner, andproliferation can be inhibited by an hIL-23-neutralizing agent. In theassay, SEQ ID NO: 2 was preincubated at various concentrations with aconstant amount of hIL-23, and the mixtures were subsequently added toBa/F3 cells in culture. After three days in culture, the extent ofproliferation was assessed by quantifying the number of viable cells.This was performed using the CellTiter-Glo Luminescent Cell ViabilityAssay (Promega CAT #G7571) to measure ATP levels, which correlate withthe number of metabolically active cells. The ability of SEQ ID NO: 2 toneutralize hIL-23 was assessed by its IC50 value, i.e. the concentrationof the lipocalin mutein that leads to half-maximal inhibition of hIL-23induced proliferation.

The detailed procedure of setting up the assay is hereby described inthe following. Ba/F3 transfectants were maintained in RPMI-1640 medium,10% fetal calf serum, 0.05 mM 2-mercaptoethanol, 500 μg/mL geneticin(G418), 1 ng/mL mIL-3, 2 μg/mL puromycin, and 200 μg/mL zeocin. Ba/F3proliferation assays were carried out in RPMI-1640 medium, 10% fetalcalf serum, and 0.05 mM 2-mercaptoethanol. Assays were performed in384-well white clear flat-bottom plates (Greiner) in 25 μL per well.

On day 1, cells from a Ba/F3 suspension cell culture were counted,pelleted, washed twice in assay medium, and plated at a density of 2500cells per well. 50 pM of hIL-23 (CAT #HZ-1254, HumanZyme)—correspondingto the predetermined EC50 required to induce Ba/F3 cellproliferation—and dilution series of SEQ ID NO: 2, a negative control(human IgG isotype antibody, CAT #. 009-000-003, Dianova) and ofbenchmark antibodies (benchmark 3: SEQ ID NO: 57 and SEQ ID NO: 58,benchmark 4: SEQ ID NO: 59 and SEQ ID NO: 60) were added to the wells.All titration series were performed with a serial 1:3 dilution in assaymedium and a suitable starting concentration. Subsequently, the cellswere allowed to proliferate for 72 hours at 37° C. To ensure that thepotency of hIL-23 was not subject to inter- and intra-day variability,the dose-dependent proliferation response of the Ba/F3 cells to hIL-23was checked by adding a dilution series of hIL-23 alone to Ba/F3 cells,using 1:3 dilution steps in assay medium starting with 50 nM. Toquantify cell proliferation after 72 hours, 25 μL CellTiter-Glo reagentswere added to the cells in each of the wells and incubated for 2 minuteson an orbital shaker to induce cell lysis, and luminescence was measuredusing the PheraStar FS reader.

EC50 values were determined using GraphPad Prism software (GraphPadSoftware Inc., San Diego, Calif., USA) by plotting Luminescence signalagains samples' concentration and non-linear regression of the data witha sigmoidal dose-response model.

The result of the experiment is shown in FIG. 10. The proliferationassay disclosed above is representative of two independent experiments.SEQ ID NO: 2 displayed an average EC50 of 1.2 nM (1.7 nM in a firstexperiment, 0.7 nM in the repeat experiment), benchmark 3 exhibited anEC50 of 3.0 nM (3.1/2.9), and benchmark 4 exhibited an EC50 of 1.2 nM(0.8/1.5). The negative control had no effect on proliferation. The datatherefore demonstrates that SEQ ID NO: 2 and the benchmark moleculesexhibit a comparable potency in this functional assay.

Example 11: Design and characterization of fusion proteins

Various fusion proteins containing either one or both of the IL-17A andIL-23-binding lipocalin muteins were generated, expressed andcharacterized as follows and as depicted in FIG. 11.

SEQ ID NO: 8 is a fusion protein of SEQ ID NO: 1 and a deimmunisedalbumin binding peptide (dABD, SEQ ID NO: 15), which is derived from thealbumin binding domain of streptococcal protein G.

SEQ ID NO: 7 is a fusion protein of SEQ ID NO: 2 and SEQ ID NO: 15.

SEQ ID NO: 10 is a homodimeric fusion protein of two SEQ ID NO: 1sequences in a row, linked via linker SEQ ID NO: 18, and fused to SEQ IDNO: 15.

SEQ ID NO: 3 is a heterodimeric fusion protein of SEQ ID NO: 1 and SEQID NO: 2, linked via linker SEQ ID NO: 18; while SEQ ID NO: 4 is aheterodimeric fusion protein with a reversed lipocalin mutein order, SEQID NO: 2 and SEQ ID NO: 1, and linked with the slightly different linkerSEQ ID NO: 19.

SEQ ID NO: 5 is a fusion protein corresponding to SEQ ID NO: 3, but withan additional C-terminal fusion to the albumin binding domain ofstreptococcal protein G (SEQ ID NO: 14)

SEQ ID NO: 9 is a fusion protein corresponding to SEQ ID No: 3, but withan additional C-terminal fusion to dABD (SEQ ID NO: 15).

SEQ ID 6 is a trimeric fusion protein consisting of two SEQ ID NO: 1sequences in a row, fused C-terminally to SEQ ID NO: 2 and dABD (SEQ IDNO: 15). The linkers between the lipocalins consisted of SEQ ID NO: 18.

We also generated fusion proteins of one or more SEQ ID NO: 1 moeitiesto the Fc part of IgG1, which corresponds to SEQ ID NO: 16:

SEQ ID NO: 11 is a fusion protein corresponding to a C-terminal fusionof SEQ ID NO: 1 to SEQ ID NO: 16, linked via the peptide linker SEQ IDNO: 19.

SEQ ID NO: 12 is a fusion protein corresponding to a N- and C-terminaldouble fusion of SEQ ID NO: 1, such that the Fc molecule of SEQ ID NO:16 is endowed with SEQ ID NO: 1 at both the N- and the C-termini.

SEQ ID NO: 13 is a fusion protein corresponding to a C-terminal fusionof the homodimeric fusion protein of SEQ ID NO: 10 to the Fc molecule ofSEQ ID NO: 16.

All fusion proteins were—where applicable—fully characterized by assaysexactly as described above for SEQ ID NO: 1 and SEQ ID NO: 2:competition ELISA assays for hIL-17A (Example 3) and hIL-23 (Example 8),SPR assays where the fusion protein was immobilized on an SPR chip forhIL-17AF (Example 2) and hIL-23 (Example 6), and functional cell-basedassays as described for hIL-17A (Example 5) and hIL-23 (Example 10).

The experimental results are summarized in Table 1. The table providesan overview of the activity of individual ipocalin muteins SEQ ID NO: 1and SEQ ID NO: 2, compared to their fusion proteins SEQ ID NOs: 3-13 incompetition ELISA, surface plasmon resonance (SPR) and functionalcell-based assays. Values were determined for the interaction with IL-17and/or IL-23, depending on whether the respective construct contains theIL-17A-binding lipocalin mutein SEQ ID NO: 1, the IL-23-bindinglipocalin mutein SEQ ID NO: 2, or both. To determine the activitytowards IL-17 and IL-23, respectively, competition ELISA experimentswere carried out as described in Example 3 and/or Example 8, SPRexperiment in reverse format—i.e. with the protein constructsimmobilized on the sensor chip—were carried out as described in Example2 and/or Example 6, and cell assays were based on either IL-17A-inducedG-CSF secretion (Example 5) and/or IL-23 induced Ba/F3 cellproliferation (Example 10). Note that the reverse format SPR experimentperformed to determine IL-23 affinity was carried out in the presence ofunphysiologically high concentrations of NaCl, and that the valuestherefore do not reflect the affinity to IL-23 under physiologicalconditions, but serve to determine whether the relative affinity toIL-23 is different in the fusion proteins SEQ ID NO's: 3-13 compared tothe individual muteins SEQ ID NO: 1 and SEQ ID NO: 2. Table 1demonstrates that the IL-17A-binding activity of all fusions containingSEQ ID NO: 1 is at least as good as that of SEQ ID NO: 1 itself in allassay formats. SEQ ID NO: 1 can therefore flexibly be employed in anyfusion protein without activity loss. The IL-23-binding activity of allfusions containing SEQ ID NO: 2 is very close to that of SEQ ID NO: 2itself in all assay formats. SEQ ID NO: 2 can therefore flexibly beemployed in any fusion protein without significant activity loss.

Example 12: An SPR Experiment Designed to Show Simultaneous Binding ofall Targets by Fusion Protein

To demonstrate simultaneous binding of the fusion protein of SEQ ID NO:9 to hIL-17A, hIL-23 and human serum albumin (HSA), a surface plasmonresonance (SPR) based assay was employed using a Biacore T200 instrument(GE Healthcare). SEQ ID NO: 9 was biotinylated as described in Example2. In the SPR affinity assay, biotinylated SEQ ID NO: 9 was captured ona sensor chip CAP using the Biotin CAPture Kit (GE Healthcare). To thisend, undiluted Biotin CAPture Reagent was applied at a flow rate of 2μL/min for 300 s. Subsequently, 0.4 to 10 μg/mL of biotinylated SEQ IDNO: 9 was applied for 300 s at a flow rate of 5 μL/min. The referencechannel was loaded with Biotin CAPture Reagent only.

To demonstrate simultaneous binding, dilutions of hIL-17 A/F, hIL-23 andHSA (200 nM, 1000 nM and 2000 nM) were prepared in HBS-EP+ buffersupplemented with 350 mM NaCl and consecutively applied to the preparedchip surface. Applying a flow rate of 30 μL/min, hIL-17 A/F, hIL-23 andHSA were consecutively injected with a sample contact time of 300 s. Theapplication of target to immobilized SEQ ID NO: 9 was also performedemploying the single ligands hIL-17 A/F, hIL-23 and HSA, to obtain themaximum binding levels obtainable by binding a single target forcomparison.

FIG. 12 compares the measured binding curve with a theoretical bindingcurve reflecting complete binding of all ligands. The latter wasobtained by assembling the experimental response of SEQ ID NO: 9 to theindividual ligands. The measured and the theoretical curve are nearlyidentical, with the exhibited difference attributable to dissociation ofthe targets in the experimental curve. The data shows that SEQ ID NO: 9is capable of simultaneously binding all targets, hIL-17A, hIL-23 andHSA, without a loss of signal intensity or a change in kinetics comparedto binding a single target only.

Example 13: Affinity of Alternative Lipocalin Muteins to IL-23

To measure the binding affinity of the lipocalin muteins SEQ ID NO: 45and SEQ ID NO: 46 to human IL-23, a Surface Plasmon Resonance (SPR)based assay was employed utilizing a Biacore T200 instrument (GEHealthcare). In the SPR affinity assay (FIG. 13), hIL-23 was immobilizedon a sensor chip using standard amine chemistry: Activation of the chip,immobilization of hIL-23, SPR measurements and data evaluation wereperformed as described in Example 6.

As shown in FIG. 13, the resulting fit curves demonstrate that SEQ IDNO: 45 bound with high affinity to hIL-23, with an association rateconstant of k_(a)=3.0×10⁵ M⁻¹sec⁻¹ and a dissociation rate constant ofk_(d)=3×10⁻⁵ sec⁻¹, resulting in a dissocation constant of K_(D)=100 pM.Similarly, as shown in FIG. 13, SEQ ID NO: 46 bound with high affinityto hIL-23, with an association rate constant of ka=7.0×10⁴ M⁻¹sec⁻¹ anda dissociation rate constant of kd=4.0×10⁻⁵ sec⁻¹, resulting in adissocation constant of K_(D)=0.6 nM.

Example 14: Competitive Mode of Action of Lipocalin Muteins to IL-23

Whether the lipocalin muteins SEQ ID NO: 45 and SEQ ID NO: 46 bind tohuman IL-23 in a competitive mode was tested in vitro using acompetition ELISA format (FIG. 14). The experiment and evaluation werecarried out in an identical fashion compared to Example 8.

The result of the experiment is shown in FIG. 14. SEQ ID NO: 45 exhibitsa competitive binding to hIL23, with a fitted IC50 value of 0.1 nM, andSEQ ID NO: 46 also displays a competitive binding to hIL23, with afitted IC50 value of 1.1 nM.

Example 15: Lipocalin-Mutein-Mediated Blockade of IL-23 in Cell-BasedProliferation Assays

The ability of the lipocalin muteins SEQ ID NO: 45 and SEQ ID NO: 46 toneutralize the biological activity of hIL-23 was assessed by theapplication of short-term proliferation bioassays employing cells thatrecombinantly express the human IL-23 receptor. The experiment andevaluation were carried out in analogy to Example 10. SEQ ID NO: 43served as the negative control.

The result of the experiment is shown in FIG. 15. SEQ ID NO: 45 displaysan average EC50 of 3.7 nM, and SEQ ID NO: 46 exhibits an EC50 of 5.4 nM.The negative control had no effect on proliferation. The data thereforedemonstrates that SEQ ID NO: 2 and the lipocalin muteins of SEQ ID NO:45 and of SEQ ID NO: 46 exhibit a comparable potency in this functionalassay.

Example 16: Specificity of Fusion Protein Towards IL-17A

We employed an ELISA assay to determine the specificity of the fusionprotein of SEQ ID NOs: 63 and 62 to IL-17A. Neutravidin was dissolved inPBS (5 μg/mL) and coated overnight on microtiter plates at 4° C. Theplate was washed after each incubation step with 100 μL PBS supplementedwith 0.1% (v/v) Tween 20 (PBS-T) five times. The plates were blockedwith 2% BSA (w/v) in PBS-T (PBS-TB) for 1 h at room temperature andsubsequently washed. IL-17A (Peprotech) which had been biotinylated wascaptured on neutravidin for 20 min at a concentration of 1 μg/ml.Unbound protein was washed off. Subsequently, different concentrationsof the lipocalin mutein of SEQ ID NO: 1 or the fusion protein were addedto the wells and incubated for 1 h at room temperature, followed by awash step. Bound fusion protein or lipocalin mutein were detected afterincubation with 1:2000 diluted anti-human TLc antibody conjugated to HRPin PBS-TB. After an additional wash step, fluorogenic HRP substrate(QuantaBlu, Thermo) was added to each well and the fluorescenceintensity was detected using a fluorescence microplate reader.

The result of the experiment is depicted in FIG. 16, together with thefit curves resulting from a 1:1 binding sigmoidal fit, where the EC50value and the maximum signal were free parameters, and the slope wasfixed to unity. The resulting EC50 values are provided in Table 2,including the errors of the sigmoidal fit of the data. The observed EC50values are, within the errors of the experiment, very similar for theantibody-lipocalin mutein fusion protein and the lipocalin mutein. Theexperiment shows that the lipocalin mutein of SEQ ID NO: 1 as includedin the fusion protein can be fused to the antibody of SEQ ID NO: 61 and62 without a loss in activity towards IL-17A.

TABLE 2 ELISA data for IL-17A binding EC50 IL-17A Name [nM] SEQ ID NO: 11.14 ± 0.10 SEQ ID NOs: 63 and 62 1.25 ± 0.17

Example 17: Specificity of Fusion Protein Towards IL-23

We employed an ELISA assay to determine the specificity of the fusionprotein of SEQ ID NOs: 64 and 62 towards IL-23. Neutravidin wasdissolved in PBS (5 μg/mL) and coated overnight on microtiter plates at4° C. The plate was washed after each incubation step with 100 μL PBSsupplemented with 0.1% (v/v) Tween 20 (PBS-T) five times. The plateswere blocked with 2% BSA (w/v) in PBS-T for 1 h at room temperature andsubsequently washed. Biotinylated IL-23 was captured on neutravidin for20 min at a concentration of 1 μg/ml. Unbound protein was washed off.Subsequently, different concentrations of the lipocalin mutein of SEQ IDNO: 2 or the fusion proteins were added to the wells and incubated for 1h at room temperature, followed by a wash step. Bound fusion proteins orlipocalin mutein were detected after incubation with 1:1000 dilutedanti-human NGAL antibody conjugated to HRP in PBS-T supplemented with 2%(w/v) BSA (PBS-TB). After an additional wash step, fluorogenic HRPsubstrate (QuantaBlu, Thermo) was added to each well and thefluorescence intensity was detected using a fluorescence microplatereader.

The result of the experiment is depicted in FIG. 17, together with thefit curves resulting from a 1:1 binding sigmoidal fit, where the EC50value and the maximum signal were free parameters, and the slope wasfixed to unity. The resulting EC50 values are provided in Table 3. Theobserved EC50 values for the antibody-lipocalin mutein fusion proteinand the lipocalin mutein are very similar within the error of theexperiment. This demonstrates that the lipocalin mutein of SEQ ID NO: 2as included in the fusion protein can be fused to the antibody of SEQ IDNO: 64 and 62 without a loss in activity towards IL-23.

TABLE 3 ELISA data for IL-23 binding EC50 IL-23 Name [nM] SEQ ID NO: 21.3 ± 0.08 SEQ ID NOs: 64 and 62 1.2 ± 0.08

Example 18: Specificity of Fusion Proteins Towards TNF-α

We employed an ELISA assay to determine the specificity of the fusionprotein of SEQ ID NOs: 63 and 62 and the fusion protein of SEQ ID NOs:64 and 62 to TNF-α. The antibody of SEQ ID NO: 61 and 62 served as thepositive control. Recombinant TNF-α (R&D Systems, 210-TA-100/CF) wasdissolved in PBS (1 μg/mL) and coated overnight on microtiter plates at4° C. The plate was washed five times after each incubation step with100 μL PBS-T. The plates were blocked with 2% BSA (w/v) in PBS-T for 1 hat room temperature and subsequently washed. Different concentrations ofthe TNFα-specific parental antibody or the fusion proteins were added tothe wells and incubated for 1 h at room temperature, followed by a washstep. Bound fusion proteins or antibody were detected after incubationfor 1 h at room temperature with 1:5000 diluted goat anti-human IgG Fabantibody conjugated to HRP (Jackson Laboratories) in PBS-TB. After anadditional wash step, fluorogenic HRP substrate (QuantaBlu, Thermo) wasadded to each well and the fluorescence intensity was detected using afluorescence microplate reader.

The result of the experiment is depicted in FIG. 18, together with thefit curves resulting from a 1:1 binding sigmoidal fit, where the EC50value and the maximum signal were free parameters, and the slope wasfixed to unity. The resulting EC50 values are provided in Table 4. Theobserved EC50 values for all proteins tested are very similar. Thisdemonstrates that the antibody as included in the fusion proteins can befused to different lipocalin muteins without compromising its activitytowards TNFα.

TABLE 4 ELISA data for TNFα binding EC50 TNFα Name [nM] SEQ ID NOs: 61and 62 0.16 ± 0.01 SEQ ID NOs: 63 and 62 0.22 ± 0.01 SEQ ID NOs: 64 and62 0.26 ± 0.01

Example 19: Demonstration of Simultaneous Target Binding of FusionProteins in an ELISA-Based Setting

In order to demonstrate the simultaneous binding of the fusion proteinof SEQ ID NOs: 63 and 62 and the fusion protein of SEQ ID NOs: 64 and 62to TNFα and IL-17A or IL-23, respectively, a dual-binding ELISA formatwas used. Recombinant TNF-α (R&D Systems, 210-TA-100/CF) in PBS (1μg/mL) was coated overnight on microtiter plates at 4° C. The plate waswashed five times after each incubation step with 100 μL PBS-T. Theplates were blocked with 2% BSA (w/v) in PBS-T for 1 h at roomtemperature and subsequently washed again. Different concentrations ofthe fusion proteins were added to the wells and incubated for 1 h atroom temperature, followed by a wash step. Subsequently, biotinylatedIL-17A (in case of SEQ ID NOs: 63 and 62) or biotinylated IL-23 (in caseof SEQ ID NOs: 64 and 62) were added at a constant concentration of 1μg/mL in PBS-TB for 1 h. After washing, Extravidin-HRP (Sigma-Adrich,1:5000 in PBS-TB) was added to the wells for 1 h. After an additionalwash step, fluorogenic HRP substrate (QuantaBlu, Thermo) was added toeach well and the fluorescence intensity was detected using afluorescence microplate reader.

The result of the experiment is depicted in FIG. 19, together with thefit curves resulting from a 1:1 binding sigmoidal fit, where the EC50value and the maximum signal were free parameters, and the slope wasfixed to unity. The resulting EC50 values are provided in Table 5. Allfusion proteins showed clear binding signals with EC50 values in thesingle digit nanomolar range, demonstrating that the fusion proteins areable to engage TNFα and either IL-17A or IL-23 simultaneously.

TABLE 5 ELISA data for simultaneous target binding EC50 Dual bindingName [nM] SEQ ID NOs: 63 and 62 2.70 ± 0.22 SEQ ID NOs: 64 and 62 1.54 ±0.16

Embodiments illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including”, “containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention. Thus, it should beunderstood that although the present embodiments have been specificallydisclosed by preferred embodiments and optional features, modificationand variations thereof may be resorted to by those skilled in the art,and that such modifications and variations are considered to be withinthe scope of the invention. All patents, patent applications, textbooksand peer-reviewed publications described herein are hereby incorporatedby reference in their entirety. Furthermore, where a definition or useof a term in a reference, which is incorporated by reference herein isinconsistent or contrary to the definition of that term provided herein,the definition of that term provided herein applies and the definitionof that term in the reference does not apply. Each of the narrowerspecies and subgeneric groupings falling within the generic disclosurealso forms part of the invention. This includes the generic descriptionof the invention with a proviso or negative limitation removing anysubject matter from the genus, regardless of whether or not the excisedmaterial is specifically recited herein. In addition, where features aredescribed in terms of Markush groups, those skilled in the art willrecognize that the disclosure is also thereby described in terms of anyindividual member or subgroup of members of the Markush group. Furtherembodiments will become apparent from the following claims.

Equivalents: Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims. Allpublications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

1-96. (canceled)
 97. A polypeptide having binding specificity forIL-23p19, wherein the polypeptide comprises a lipocalin mutein thatbinds IL-23p19 with a K_(D) of about 1 nM or lower.
 98. A polypeptidehaving binding specificity for IL-23p19, wherein the polypeptidecomprises a lipocalin mutein that binds IL-23p19 with a detectableaffinity, wherein the lipocalin mutein comprises one or more of thefollowing mutated amino acid residues at the sequence positions 28, 36,40-41, 49, 52, 68, 70, 72-73, 77, 79, 81, 87, 96, 100, 103, 106,124-125, 127, 132 and 134 of the linear polypeptide sequence of themature human NGAL (SEQ ID NO: 43): Gln 28→His; Leu 36→Glu; Ala 40→Leu;Ile 41→Leu; Gln 49→Arg; Tyr 52→Thr; Asn 65→Asp; Ser 68→Arg; Leu 70→Glu;Arg 72→Gly; Lys 73→Ala or Vla; Lys 75→Thr; Asp 77→Lys; Trp 79→Gln orArg; Arg 81→Gly; Asn 96→Gly; Lys 98→Giu; Tyr 100→Met; Leu 103→Met; Tyr106→Phe; Asn 114→Asp; Met 120→Ile; Lys 125→Tyr; Ser 127→Tyr; and Lys134→Glu.
 99. The polypeptide of claim 97, wherein said lipocalin muteinis crossreactive with both human IL-23 and mouse IL-23.
 100. Thepolypeptide of claim 97, wherein the lipocalin mutein comprises one ofthe following sets of amino acid substitutions in comparison with thelinear polypeptide sequence of the mature human NGAL (SEQ ID NO: 43):(1) Gln 28→His; Leu 36→Glu; Ala 40→Leu; Ile 41→Leu; Gln 49→Arg; Tyr52→Thr; Asn 65→Asp; Ser 68→Arg; Leu 70→Glu; Arg 72→Gly; Lys 73→-3-Ala;Lys 75→Thr; Cys 76→Tyr; Asp 77→Lys; Trp 79→Gln; Arg 81→Gly; Asn 96→Gly;Lys 98→Glu; Tyr 100→Met; Leu 103→Met; Tyr 106→Phe; Met 120→Ile; Lys125→Tyr; Ser 127→Tyr; and Lys 134→Glu; (2) Gln 28→His; Leu 36→Glu; Ala40→Leu; Gln 49→Arg; Tyr 52→Thr; Asn 65→Asp; Ser 68→Arg; Leu 70→Glu; Arg72→Gly; Lys 73→Vla; Lys 75→Thr; Cys 76→Arg; Asp 77→Lys; Trp 79→Arg; Arg81→Gly; Asn 96→Gly; Tyr 100→Met; Leu 103→Met; Tyr 106→Phe; Lys 125→Tyr;Ser 127→Tyr; and Lys 134→Glu; or (3) Gln 28→His; Leu 36→Glu; Ala 40→Leu;Ile 41→Leu; Gln 49→Arg; Tyr 52→Thr; Asn 65→Asp; Ser 68→Arg; Leu 70→Glu;Arg 72→Gly; Lys 73→Val; Lys 75→Thr; Cys 76→Tyr; Asp 77→Lys; Trp 79→Gln;Arg 81→Gly; Asn 96→Gly; Tyr 100→Met; Leu 103→Met; Tyr 106→Phe; Asn114→Asp; Lys 125→Tyr; Ser 127→Tyr; and Lys 134→Glu.
 101. The polypeptideof claim 97, wherein the lipocalin mutein has at least 70% identity toan amino acid sequence selected from the group consisting of SEQ ID NOs:2 and 45-46.
 102. A composition comprising a polypeptide of claim 97.103. A method of detecting the presence of IL-23p19 in a sample, themethod comprising contacting the sample with a polypeptide of claim 97,under conditions that allow the formation of complex of the polypeptideand IL-23p19.
 104. A method of binding IL-23p19 in a subject, comprisingadministering to said subject an effective amount of one or morepolypeptides of claim 97, or of one or more compositions comprising suchpolypeptides.
 105. A method for inhibiting the binding of IL-23 to itsreceptor in a subject, comprising administering to said subject aneffective amount of one or more polypeptides of claim 97, or of one ormore compositions comprising such polypeptides.
 106. A kit comprising atleast one polypeptide of claim 97, and one or more instructions forusing the kit.
 107. A nucleic acid molecule comprising a nucleotidesequence encoding a polypeptide of claim
 97. 108. A host cell containinga nucleic acid molecule of claim
 107. 109. A method of producing apolypeptide of claim 97, wherein the polypeptide is produced startingfrom the nucleic acid coding for the polypeptide by means of geneticengineering methods.